Selection and use of umbilical cord cell fractions suitable for transplantation

ABSTRACT

Methods of selecting umbilical cord blood units for ex-vivo expansion, separation of CD133+/CD34+ positive and uncultured CD133+/CD34+ negative fractions, methods for expanding the selected CD133+/CD34+ fraction, selection of expanded populations of CD133+/CD34+ cord blood cells for transplantation to subjects in need thereof and the therapeutic use of suitable selected, ex-vivo expanded CD133+/CD34+ and unselected CD133/CD34 negative cord blood fractions for transplantation in the clinical setting, for treatment of hematological malignancies are provided. The present invention also envisions kits comprising the expanded and unselected cord blood fractions.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of selecting umbilical cordblood units for ex-vivo expansion, separation of CD133+/CD34+ positiveand uncultured CD133+/CD34+ negative fractions, methods for expandingthe selected CD133+/CD34+ fraction, selection of expanded populations ofCD133+/CD34+ cord blood cells for transplantation to subjects in needthereof and the therapeutic use of suitable selected, ex-vivo expandedCD133+/CD34+ and unselected CD133/CD34 negative cord blood fractions fortransplantation in the clinical setting, for treatment of hematologicalmalignancies. The present invention also envisions kits comprising theexpanded and unselected cord blood fractions.

More than two thirds of patients awaiting hematopoietic stem cell istransplantation lack a suitable matched related donor, making unrelateddonor grafts a necessity. Cord blood, bone marrow and otherhematopoietic-rich tissues are a valuable source of stem cells,particularly where a matched unrelated donor cannot be found within areasonable time. Advantages of the use of cord blood include the factthat it is readily available, carries less risk of transmission ofblood-borne infectious diseases, and is transplantable across HLAbarriers with diminished risk of graft-versus-host disease compared withsimilarly mismatched stem cells from the peripheral blood or bone marrowof related or unrelated donors. Another important advantage of cordblood is the rapidity with which an acceptable cord-blood unit, onceidentified, can be acquired.

However, a major clinical limitation of umbilical cord blood is the lowstem cell dose available for transplantation, compared to mobilizedperipheral blood (PB) or bone marrow. This low stem cell dose cancompromise the chances of engraftment and contributes to delayedkinetics of neutrophil and platelet recovery, as well as othertransplant outcomes, resulting in transplant related complications,morbidity and mortality, and longer duration of hospitalization. Toaddress this shortcoming, several approaches have been developed,including dual umbilical cord blood transplantation (DCBT) and ex vivoexpansion of cord blood stem cells. DCBT has become standard practice incord blood transplantation for recipients in whom a single cord bloodunit of adequate cell dose is unavailable.

Still, engraftment kinetics of DCBT are no better than single unittransplants. Ex vivo expansion is still an experimental approach. Theaim of ex vivo expansion of cord blood is to provide a graft withsufficient numbers of cells that have rapid and robust in vivo neutophiland platelet producing potential to enable successful transplantation.Delaney et al. (2010. Nat Med 16:232-236) and De Lima et al. (2012, NEngl J Med, 367:2305-2315) published the results of two clinical studiesemploying ex vivo expanded cord blood grafts in a double cordconfiguration, i.e., one unit used for expansion, and a second unitadministered unmanipulated, reporting that although the expanded cellswere observed as early as one week post-transplantation, they weremostly lost before or after engraftment, while the unmanipulated unitpredominated in all of the recipients.

The present inventors have shown that culturing CD133+ cord blood cellsin the presence of cytokines (SCF, TPO, IL-6 and FLT-3 ligand) andnicotinamide resulted in better and prolonged expansion of both late andearly progenitors, which are important for short-term early trilineageengraftment, and reduction in the fraction of differentiatedmyelomonocitic cells (CD14+, CD11b+, CD11c+) and increase the fractionof less differentiated early progenitor cells, the CD34+CD38− cells.CD34+ cells obtained following culturing with NAM displayed increasedmigration towards SDF-1 and home to the bone marrow (24 hour postinfusion) with higher efficacy than cells cultured with cytokines onlyor non-cultured cells. Further, in vitro studies show that these cultureconditions do not support expansion of T cells and therefore onlynegligible amounts of cells displaying T-cell phenotype can be found inthe expanded cord blood fraction (see U.S. Pat. Nos. 7,955,852 and8,846,393, and Peled et al, 2012, Exp Hematol 40:342-55).

Horowitz et al (2014, J Clin Invest 124:3121-3128), using the doublecord blood transplantation approach, demonstrated that allogeneic stemand progenitor cells ex-vivo expanded from an entire cord blood unitwith cytokines and nicotinamide are capable of out-competing anunmanipulated cord blood graft, and providing rapid engraftment androbust, long term multilineage hematopoiesis.

Wagner et al (Cell Stem Cell 2016 18:144-55) reported positive resultsin a Phase I/II clinical trial with cord blood hematopoietic stem cellsexpanded with an aryl hydrocarbon receptor antagonist as a stand alonegraft.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of selecting a cryopreserved umbilical cordblood unit for ex-vivo expansion and transplantation into a subject,comprising:

-   -   (a) determining in the umbilical cord blood unit prior to        ex-vivo expansion the following pre-cryopreservation parameters:        -   (i) about 8×10⁶ to about 15×10⁶ total CD34+ cells;        -   (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A            and HLA-B, low resolution) and HLA class II (HLA-DRB1, high            resolution) loci with the subject;        -   (iii) about 1.8×10⁹ to about 3.0×10⁹ pre-cryopreserved total            nucleated cells;        -   (iv) about 1.5×10⁷ to about 3.0×10⁷ pre-cryopreserved total            nucleated cells per kilogram subject weight,    -   (b) selecting or excluding the umbilical cord blood unit        according to the parameters, thereby selecting an umbilical cord        blood unit suitable for ex-vivo expansion and transplantation        into the subject.

According to an aspect of some embodiments of the present invention (i)is about 8×10⁶ to about 10×10⁶ total CD34+ cells.

According to an aspect of some embodiments of the present invention (i)is at least 8×10⁶ total CD34+ cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a hematological disease in asubject in need thereof, the method comprising:

-   -   (a) separating a single umbilical cord blood unit suitable for        transplantation into the subject into (i) a first, selected        blood cell fraction comprising CD133+/CD34+ selected cells        and (ii) a second, unselected blood cell fraction comprising        CD133/CD34 negative cells;    -   (b) ex vivo culturing the first blood cell fraction comprising        CD133+/CD34+ selected cells under conditions allowing for cell        proliferation, the conditions comprising providing nutrients,        serum and a combination of cytokines including each of stem cell        factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in        an amount between 1.0 mM to 10 mM;    -   (c) cryopreserving the ex-vivo cultured first. CD133+/CD34+        selected blood cell fraction and the uncultured second,        unselected blood cell fraction from step (b),    -   (d) thawing the ex-vivo cultured first selected and the        uncultured second, unselected blood cell fractions, and    -   (e) transplanting the thawed ex-vivo cultured first selected and        uncultured second unselected blood cell fractions from step (d)        into a subject in need thereof,    -   thereby treating the hematological disease in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a hematological disease in asubject in need thereof, the method consisting of:

-   -   (a) separating a single umbilical cord blood unit suitable for        transplantation into the subject into (i) a first, selected        blood cell fraction comprising CD133+/CD34+ selected cells        and (ii) a second, unselected blood cell fraction comprising        CD133/CD34 negative cells;    -   (b) ex vivo culturing the first blood cell fraction comprising        CD133+/CD34+ selected cells under conditions allowing for cell        proliferation, the conditions comprising providing nutrients,        serum and a combination of cytokines including each of stem cell        factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in        an amount between 1.0 mM to 10 mM;    -   (c) cryopreserving the ex-vivo cultured first, selected        CD133+/CD34+ blood cell fraction and the uncultured second,        unselected blood cell fraction from step (b),    -   (d) thawing the ex-vivo cultured first selected and the        uncultured second, unselected blood cell fractions, and    -   (e) transplanting the thawed ex-vivo cultured first selected and        uncultured second unselected blood cell fractions from step (d)        into a subject in need thereof,    -   thereby treating the hematological disease in the subject.

According to some embodiments of the present invention the singleumbilical cord blood unit suitable for transplantation is characterizedby the following pre-cryopreservation parameters:

-   -   (i) at least 8×10⁶ total CD34+ cells;    -   (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A and        HLA-B, low resolution) and HLA class II (HLA-DRB1, high        resolution) loci with the subject;    -   (iii) about 1.8×10⁹ to about 3.0×10⁹ pre-cryopreserved total        nucleated cells, and    -   (iv) about 1.5×10⁷ to about 3.0×10⁷ pre-cryopreserved total        nucleated cells per kilogram subject weight.

According to some embodiments of the present invention the cryopreservedex-vivo cultured first selected and the uncultured second, unselectedblood cell fractions are thawed on the same day of transplantation.

According to some embodiments of the present invention the cryopreservedex-vivo cultured first selected and the uncultured second, unselectedblood cell fractions are thawed and reconstituted in infusion solution.

According to some embodiments of the present invention thetransplantation is affected by infusion in an infusion solution into thepatient.

According to some embodiments of the present invention the infusionsolution comprises 8% Human Serum Albumin (HSA) and 6.8% weight pervolume Dextran 40.

According to some embodiments of the present invention the ex-vivocultured first selected blood cell fraction is infused prior to theuncultured second, unselected blood cell fraction.

According to some embodiments of the present invention the unculturedsecond, unselected blood cell fraction is infused prior to the ex-vivocultured first selected blood cell fraction.

According to some embodiments of the present invention the first ex-vivocultured blood cell fraction comprises at least 8×10⁸ total viablecells.

According to some embodiments of the present invention the seconduncultured blood cell fraction comprises at least 4×10⁸ total viablecells.

According to some embodiments of the present invention the hematologicaldisease is a hematological malignancy.

According to some embodiments of the present invention the hematologicalmalignancy is selected from the group consisting of Chronic myelogenousleukemia (CML), acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML) and myelodysplastic syndrome (MDS).

According to some embodiments of the present invention the subject hasbeen subjected to myeloablative therapy or other conditioning regimeprior to transplantation.

According to some embodiments of the present invention the subject hasbeen to subjected to graft-versus-host disease (GvHD) prophylaxis regimeprior to transplantation.

According to some embodiments of the present invention the subject issubjected to graft-versus-host disease (GvHD) prophylaxis regimefollowing transplantation.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases time fromtransplantation to neutrophil engraftment in the subject, when comparedto transplantation of a single or double unit of unmanipulated cordblood.

According to some embodiments of the present invention the time toneutrophil engraftment is decreased 5-14 days when compared totransplantation of a single or double unit of unmanipulated cord blood.

According to some embodiments of the present invention the neutrophilengraftment consists of achieving an absolute neutrophil count (ANC)≥0.5×10⁹/L on 3 consecutive measurements on different days withsubsequent donor chimerism (≤10% host cells by peripheral bloodchimerism), on or before 42 days post transplant.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases time fromtransplantation to platelet engraftment in the subject, when compared totransplantation of a single or double unit of unmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ hematopoietic stem/progenitorblood cell fraction and the second uncultured CD133−/CD34− blood cellfraction increases the probability of platelet engraftment in thesubject at 42 days post transplantation when compared to transplantationof a single or double unit of unmanipulated cord blood.

According to some embodiments of the present invention whereintransplantation of the first ex vivo cultured CD133+/CD34+ blood cellfraction and the second CD133−/CD34− uncultured blood cell fractiondecreases risk of non-engraftment by day 42 after transplantation in thesubject, when compared to transplantation of a single or double unit ofunmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases the risk ofnon-relapse mortality after transplantation in the subject, whencompared to transplantation of a single or double unit of unmanipulatedcord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases the risk ofnon-relapse mortality at 210 days after transplantation.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases the risk oftransplantation related mortality at 1 year.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases duration ofhospitalization in the subject in the first 100 dayspost-transplantation, when compared to transplantation of a single ordouble unit of unmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases duration ofpost-transplantation hospitalization by 5-30 days.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases risk ofgrade 2/3 bacterial or invasive fungal infections post-transplantationin the subject, when compared to transplantation of a single or doubleunit of unmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases risk ofgrade 2/3 bacterial or invasive fungal infections post-transplantationdecreases risk of grade 2/3 bacterial or invasive fungal infections by100 days post-transplantation in the subject, when compared totransplantation of a single or double unit of unmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases risk ofacute graft-versus-host disease grade III-IV in the subject at 100 dayspost transplantation, when compared to transplantation of a single ordouble unit of unmanipulated cord blood.

According to some embodiments of the present invention transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and thesecond uncultured CD133−/CD34− blood cell fraction decreases risk ofsevere chronic graft-versus-host disease in the subject at 180 days posttransplantation, when compared to transplantation of a single or doubleunit of unmanipulated cord blood.

According to some embodiments of the present invention the first andsecond blood cell fractions are co-administered in conjunction with anadditional treatment for hematological disease.

According to some embodiments of the present invention the additionaltreatment is selected from the group consisting of immunosuppressivetreatment, chemotherapy and radio-therapy.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising:

-   -   (a) a packaging material and    -   (b) umbilical cord blood cell fractions comprising:    -   (i) a first blood cell fraction suitable for transplantation        into a subject in need thereof comprising CD133+/CD34+ selected        cord blood cells ex-vivo cultured under conditions allowing for        cell proliferation, the conditions comprising providing        nutrients, serum and a combination of cytokines including each        of stem cell factor, thrombopoietin. FLt3 ligand and IL-6 and        nicotinamide in an amount between 1.0 mM to 10 mM; and    -   (ii) a second, uncultured, unselected blood cell fraction        suitable for transplantation into a subject in need thereof        comprising CD133/CD34 negative cells.    -   and wherein the packaging material comprises a label or package        insert indicating that the first and second umbilical cord blood        cell populations are for treating a hematological disease in a        subject in need thereof.

According to some embodiments of the present invention the first ex-vivocultured blood cell fraction comprises at least 8×10⁸ total viablecells.

According to some embodiments of the present invention the seconduncultured blood cell fraction comprises at least 4×10⁸ total viablecells.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention relates to methods of selecting umbilical cordblood units for ex-vivo expansion, separation of CD133+/CD34+ positiveand uncultured CD133+/CD34+ negative fractions, methods for expandingthe selected CD133+/CD34+ fraction, selection of expanded populations ofCD133+/CD34+ cord blood cells for transplantation to subjects in needthereof and the therapeutic use of suitable selected, ex-vivo expandedCD133+/CD34+ and unselected CD133/CD34 negative cord blood fractions fortransplantation in the clinical setting, for treatment of hematologicalmalignancies. The present invention also envisions kits comprising theexpanded and unselected cord blood fractions.

The present invention also relates to a kit comprising the expanded cordblood unit, and directions for the use thereof in the treatment ofhematological malignancies.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways.

Allogeneic hematopoietic stem cell transplantation is a life-savingprocedure for patients with hematologic disorders; yet wide applicationof this procedure is limited by the availability of suitably HLA-matcheddonors.

Accordingly, cord blood grafts are routinely and more successfully usedin the pediatric transplantation setting (only 20% of cord blood unitsin banks could suffice for a 75 kg patient according to the recommendedthreshold cell dose). Adults receiving cord blood transplantation are athigh risk of early mortality (as high as 30-60%, day 100 mortality) andinfection due to delayed engraftment and higher rates of engraftmentfailure. Indeed, publications reviewing the differences in outcomesbetween umbilical cord blood and other graft sources consistently reporthigher non-relapse mortality following cord blood transplantation,generally attributable to inadequate hematopoietic recovery, and delayedengraftment has been deemed the single greatest barrier to successfulcord blood transplantation and the most important contributor to earlynon-relapse mortality.

A significant challenge to presently available methods for graftproduction is the ability to generate an expanded population ofcommitted hematopoietic progenitor cells without compromising thenumbers of less differentiated progenitor cells (CD34+CD38− or CD34+Lin−cells). The present invention provides an expansion technology utilizingnicotinamide (NAM) for increased bone marrow homing and engraftmentefficacy. Engraftment is a multi-step process involving directedmigration of the inoculated cells, homing to the bone marrow (BM),retention within the BM niche followed by self-renewal anddifferentiation. Engraftment efficacy following expansion is known to below due to poor homing to the bone marrow compared to fresh CD34+ cellsor reduced self-renewal owing to enhanced differentiation in culture.

The methods for the selection of cord blood units and ex vivo expansionof hematopoietic progenitor cells (HPC) provided herein permitincreasing their numbers while maintaining their self-renewal capacityand their ability to home to the bone marrow (BM) and efficientlyreconstitute hematopoiesis. Addition of nicotinamide delaysdifferentiation and increases the homing and engraftment efficacy ofcord-blood derived, purified CD133+ cells cultured with a combination of4-cytokines (FLT3, SCF, TIN) and IL6) for 21 (+/−2) days (19-23 days).As these culture conditions to not support expansion of T cells (onlynegligible amounts of cells displaying T-cell phenotype can be found inthe cultured, expanded cells), the present invention provides anuncultured, unmanipulated fraction derived from the same cord blood unitto provide T-cells.

The present invention provides criteria for selection of cord bloodunits for preparation of cultured, ex-vivo expanded CD133+/CD34+ anduncultured, CD133/CD34 negative cord blood fractions, methods for theculture and ex-vivo expansion of the CD133+/CD34+ cord blood fraction,criteria for selection of expanded CD133+/CD34+ and uncultured,CD133/CD34 negative cord blood fractions suitable for transplantationwith a high probability of effective engraftment and methods ofcryopreservation and preparation of the cultured and uncultured cordblood fractions. The present invention also provides methods for the useof expanded CD133+/CD34+ and uncultured, CD133/CD34 negative cord bloodfractions meeting the selection criteria for transplantation in theclinical setting, for treatment of malignant blood disorders.

Selection of Cord Blood Units for Ex-Vivo Expansion

According to some embodiments, the present invention provides a methodof selecting a cryopreserved umbilical cord blood unit for ex-vivoexpansion and transplantation into a subject, comprising: determining insaid umbilical cord blood unit prior to ex-vivo expansion the followingpre-cryopreservation parameters:

-   -   (i) about 8×10⁶ to about 15×10⁶ total CD34+ cells;    -   (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A and        HLA-B, low resolution) and HLA class II (HLA-DRB1, high        resolution) loci with said subject;    -   (iii) about 1.8×10⁹ to about 3.0×10⁹ pre-cryopreserved total        nucleated cells;    -   (iv) about 1.5×10⁷ to about 3.0×10⁷ pre-cryopreserved total        nucleated cells per kilogram subject weight,    -   and        selecting or excluding said umbilical cord blood unit according        to said parameters, thereby selecting an umbilical cord blood        unit suitable for ex-vivo expansion and transplantation into the        subject.

All cord blood units are procured from public banks that meet nationalapplicable regulations. Donors are screened and tested in accordancewith the relevant regulatory requirements. Cord blood banks typicallyemploy methods for efficient storage of freshly harvested umbilical cordblood, including red blood cell depletion and plasma depletion,resulting in volume reduction, and cryopreservation of the reducedvolume cord blood unit. Prior to cryopreservation of the cord bloodunit, a sample of the unit is analyzed to produce a profile of the unit,including, but not limited to, cell viability, amounts of specific cellpopulations, human leukocyte antigen (HLA) typing, and safety criteriasuch as bacterial/viral contamination, and unit volume, providing, interalia, total values for the entire unit, relative values (e.g.,percentage of any specific cell type from the total number of cells),and values adjusted for volume (e.g., concentration of any specific celltype in a unit volume). As used herein, the term “cryopreservation”refers to a process wherein biological entities subject to damage byunregulated chemical kinetics (e.g., cells, tissues, organelles) arepreserved by cooling to extremely low temperatures, typically −80° C.with solid carbon dioxide or −196° C. using liquid nitrogen. In oneembodiment, cord blood cells are cryopreserved with liquid nitrogen.Methods of preservation of hematopoietic cell populations are well knownin the art (see, Watts et al. Cryopreservation and Freeze-DryingProtocols, in Methods in Molecular Biology, 2007; 368:237-259).

As used herein, the term “pre-cryopreservation” refers to parameters,values or characteristics recorded prior to cryopreservation, forexample, prior to cryopreservation of a banked, cryopreserved cord bloodunit. In some embodiments, such “pre-cryopreservation” data can bereferred to for the selection of candidate cord blood units for use inthe methods of the present invention.

Thus, according to an embodiment of the present invention,pre-cryopreserved parameters of total CD34+ cells, total nucleatedcells, and red blood cell depletion and volume reduction are determinedfor an umbilical cord blood unit in order to select or exclude the cordblood unit for ex-vivo expansion and transplantation. When the identityof the candidate for transplantation (e.g., the “subject”) is known,parameters such as HLA-match (compatibility) and pre-cryopreservationtotal nucleated cells per kilogram subject weight can also be determinedand serve as a selection criteria.

In particular embodiments, cord blood units selected for ex-vivoexpansion have pre-cryopreservation total CD34+ values of about 8×10⁶ toabout 15×10⁶ total CD34+ cells, about 9×10⁶ to about 13×10⁶ total CD34+cells, about 10×10⁶ to about 12×10⁶ total CD34+ cells. In oneembodiment, cord blood units selected for ex-vivo expansion have atleast 12×10⁶ total pre-cryopreserved CD34+ cells. In one embodiment,cord blood units selected for ex-vivo expansion have at least 8×10⁶total pre-cryopreserved CD34+ cells. In one embodiment, cord blood unitsselected for ex-vivo expansion have at least 10×10⁶ totalpre-cryopreserved CD34+ cells.

In particular embodiments, cord blood units selected for ex-vivoexpansion have pre-cryopreservation total nucleated cell values of about1.8×10⁹ to about 3.0×10⁹ total nucleated cells, about 2×10⁹ to about2.7×10⁹ total nucleated cells or about 2.2×10⁹ to about 2.5×10⁹ totalnucleated cells. In one embodiment, cord blood units selected forex-vivo expansion have at least 2.5×10⁹ pre-cryopreserved totalnucleated cells. In one embodiment, cord blood units selected forex-vivo expansion have at least 2.0×10⁹ pre-cryopreserved totalnucleated cells. In one embodiment, cord blood units selected forex-vivo expansion have at least 1.8×10⁹ pre-cryopreserved totalnucleated cells. Typically, nucleated cells can be counted manually inhaemocytometers (e.g., Neubauer) or with an automated counter.

In particular embodiments, cord blood units selected for ex-vivoexpansion have pre-cryopreservation total nucleated cells per kilogramsubject weight of about 1.5×10⁷ to about 3.0×10⁷ total nucleated cellsper kilogram subject weight, about 1.7×10⁷ to about 2.8×10⁷ totalnucleated cells per kilogram subject weight, about 1.9×10⁷ to about 32.5×10⁷ total nucleated cells per kilogram subject weight or about 2×10⁷to about 2.2×10⁷ total nucleated cells per kilogram subject weight. Inone embodiment, cord blood units selected for ex-vivo expansion have atleast 3.0×10⁷ pre-cryopreserved total nucleated cells per kilogramsubject weight. In one embodiment, cord blood units selected for ex-vivoexpansion have at least 2.3×10⁷ pre-cryopreserved total nucleated cellsper kilogram subject weight. In one embodiment, cord blood unitsselected for ex-vivo expansion have at least 1.5×10⁷ pre-cryopreservedtotal nucleated cells per kilogram subject weight.

In particular embodiments, cords blood units selected for ex-vivoexpansion are pre-cryopreservation HLA-matched at at least 4 out of 6HLA class I (HLA-A and HLA-B, low resolution) and HLA class II(HLA-DRB1, high resolution) loci with said subject, at at least 5 out of6 HLA class I (HLA-A and HLA-B, low resolution) and HLA class II(HLA-DRB1, high resolution) loci with said subject, or at at 6 out of 6HLA class I (HLA-A and HLA-B, low resolution) and HLA class II(HLA-DRB1, high resolution) loci with said subject. In some embodiments,the cord units are pre-cryopreservation HLA-matched at at least 4 out of6 HLA class I (HLA-A and HLA-B, low resolution) and HLA class II(HLA-DRB1, high resolution) loci with said subject, with at least oneHLA-DRB1 match. In some embodiments, the cord units arepre-cryopreservation HLA-matched at at least 5 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject. In some embodiments, the cord unitsare pre-cryopreservation HLA-matched at at least 5 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject, with at least one HLA-DRB1 match.Typically, Class I HLA (or Major Histocompatibility Complex, MHC)antigens are determined on the cord blood cells by a microcytotoxicityassay using alloantisera for specific HLAs, complement for cytotoxicityand a dye to identify killed cells. HLA Class II are typicallydetermined by the mixed lymphocyte reaction (MLR), measuring lymphocyteproliferation following culture of mixed lymphocyte populations. HLA DRantigens can be identified by B cell antisera in a microcytotoxicityassay with enriched B cells. Antisera can be replaced by specificmonoclonal antibodies.

In some embodiments, the cords blood units selected for ex-vivoexpansion have at least 8×10⁶ total pre-cryopreserved CD34+ cells and atleast 1.5×10⁷ pre-cryopreserved total nucleated cells per kilogramsubject weight.

In some embodiments, the cords blood units selected for ex-vivoexpansion have at least 10×10⁶ total pre-cryopreserved CD34+ cells andat least 2.5×10⁷ pre-cryopreserved total nucleated cells per kilogramsubject weight.

Examples of exemplary pre-cryopreservation criteria for selection ofcord blood units for ex-vivo expansion and transplantation into asubject are presented in Tables I-IV hereinbelow.

TABLE I CBU Pre-cryopreservation selection criteria Parameter ValueTotal CD34+ cells At least 12 × 10⁶ HLA compatibility HLA- matched at atleast 4 out of 6 HLA class I (HLA-A and HLA- B, low resolution) and HLAclass II (HLA-DRB1, high resolution) loci with said subject TotalNucleated Cells (TNC) At least 1.8 × 10⁹ Total Nucleated Cells per Atleast 2.5 × 10⁷ kilogram subject weight

TABLE II CBU Pre-cryopreservation selection criteria Parameter ValueTotal CD34+ cells At least 10 × 10⁶ HLA compatibility HLA- matched at atleast 5out of 6 HLA class I (HLA-A and HLA- B, low resolution) and HLAclass II (HLA-DRB1, high resolution) loci with said subject TotalNucleated Cells (TNC) At least 2.7 × 10⁹ Total Nucleated Cells per Atleast 2.0 × 10⁷ kilogram subject weight

TABLE III CBU Pre-cryopreservation selection criteria Parameter ValueTotal CD34+ cells At least 8 × 10⁶ HLA compatibility HLA- matched at atleast 4 out of 6 HLA class I (HLA- A and HLA-B, low resolution) and HLAclass II (HLA-DRB1, high resolution) loci with said subject TotalNucleated Cells (TNC) At least 1.8 × 10⁹ Total Nucleated Cells per Atleast 1.5 × 10⁷ kilogram subject weight

TABLE IV CBU Pre-cryopreservation selection criteria Parameter ValueTotal CD34+ cells At least 13 × 10⁶ HLA compatibility HLA- matched at atleast 4 out of 6 HLA class I (HLA-A and HLA- B, low resolution) and HLAclass II (HLA-DRB1, high resolution) loci with said subject TotalNucleated Cells (TNC) At least 2.2 × 10⁹ Total Nucleated Cells per Atleast 2.7 × 10⁷ kilogram subject weight

As used herein the term “ex-vivo” refers to a process in which cells areremoved from a living organism and are propagated outside the organism(e.g., in a test tube, in a cell culture bag, etc.).

As used herein, the term “in-vitro” refers to a process in which cellsoriginating from a cell line or lines (such as embryonic cell lines,etc.) maintained in the laboratory, are manipulated outside of anorganism. Such cell lines are often immortalized cells.

As used herein, the phrase “stem cells” refers both to the lineagecommitted earliest renewable cell population responsible for generatingrapid cell mass in a tissue or body and the very early progenitor cells,which are somewhat less differentiated, not yet committed to a specificlineage and can readily revert to become a part of the long-termrenewable cell population. Hematopoietic stem cells are stem cells thatcan regenerate the cellular components of the blood, such aserythrocytes, leukocytes, platelets, etc.

As used herein, the phrases “non-stem”, “non-progenitor” and “committedcells” refer to cells at various stages of differentiation, whichgenerally no longer retain the ability to revert to become a part of arenewable cell population. Methods of ex-vivo culturing stem,progenitor, and non-stem, non-progenitor committed cells are well knownin the art of cell culturing. To this effect, see for example, the textbook “Culture of Animal Cells—A Manual of Basic Technique” by Freshney,Wiley-Liss, N. Y. (1994). Third Edition, the teachings of which arehereby incorporated by reference.

Once suitable cryopreserved cord blood units have been identified, theymay be transported for further processing (e.g., thawing and ex-vivoexpansion), or they may be marked and maintained frozen until need arisefor further processing in preparation for transplantation. Thus, a“bank” of cryopreserved cord blood units suitable for ex-vivo expansionand transplantation, and a corresponding database of the units can beestablished, enabling more rapid access to suitable cord blood units.

In some embodiments, cord blood units selected for ex-vivo expansionhave at least 1.8×10⁹ pre-cryopreserved total nucleated cells and atleast 1.5×10⁷ pre-cryopreserved total nucleated cells per kilogramsubject weight.

In some embodiments, cord blood units selected for ex-vivo expansionhave at least 2.5×10⁹ pre-cryopreserved total nucleated cells and atleast 2.0×10⁷ pre-cryopreserved total nucleated cells per kilogramsubject weight.

Further processing of cord blood units selected suitable for ex-vivoexpansion and transplantation includes thawing the selected,cryopreserved unit, and determining suitability of the thawed cord bloodcells for proceeding to further selection, separation of a CD133+/CD34+cord blood cell fraction from the CD133−/CD34− cord blood cell fraction.

Thus, according to some embodiments, there is provided a method forselecting a thawed umbilical cord blood unit for ex-vivo expansion andtransplantation into a subject, the method comprising determiningpercent viability of the cells in said thawed umbilical cord blood unitand selecting units having about 40% to about 85% viability prior toseparation of said cord blood unit into CD133+/CD34+ and CD/133−/CD34−fractions, thereby selecting a thawed umbilical cord unit suitable forex-vivo expansion and transplantation into the subject.

In some embodiments, units having about 40% to about 70% viability areselected. In some embodiments, units having about 42%, about 46%, about48%, about 50%, about 52%, about 54%, about 56%, about 58%, about 60%,about 62%, about 64%, about 66%, about 68% to about 70% viability areselected.

It will be appreciated that the thawed umbilical cord blood units aretypically cryopreserved cord blood units that have been selectedsuitable for further processing using the criteria and methods describedherein.

According to some embodiments, the selected frozen cord blood unit istransferred to the manufacturing site in accordance with the proceduresof the cord blood bank. Following confirmation that of the identity ofthe cord blood unit, the unit is stored at liquid nitrogen centralstorage until commencement of the cell selection and expansionprocedure.

At the manufacturing site, the cord blood bag is carefully wiped usingsterile alcohol wipes, thawed in a water bath at 37°±1 C and transferredto a production site (e.g. clean room) for further handling.

In some embodiments, thawed cord blood units are prepared for cellselection as follows: The thawed blood is transferred into a 175 mlcentrifuge tube. The cryobag is washed with 10% w/v dextran-40 in salinesolution and 5% w/v HSA and then added to the centrifuge tube. Twosamples are removed to determine the cell viability and viable cellconcentration of the cell suspension as well as the CD34+. CD133+content of the thawed CBU. After centrifugation the supernatant isremoved and the volume is brought to 100 ml by the addition of a PBSbuffer containing 0.4% w/v sodium citrate and 1% v/v HSA. IV Ig is addedand after incubation for 10 minutes at RT, the cell suspension iscentrifuged and the pelleted cells are re-suspended.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject and at least 8×10⁶ totalpre-cryopreserved CD34+ cells.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject and at least 2.0×10⁷pre-cryopreserved total nucleated cells per kilogram subject weight.

As used herein, the term “viability” refers to the distinction betweenliving and non-living cells. Cell viability may be judged bymorphological changes or by changes in membrane permeability and/orphysiological state inferred from the exclusion of certain dyes or theuptake and retention of others. Cell viability assays are well known inthe art, including, but not limited to trypan blue or propidium iodideexclusion and rhodamine metabolic stain (Coder. D., Current Protocols inCytometry, 1997, John Wiley and Sons, Inc., Unit 9.2, 9.2.1-9.2.14).

Thawed cord blood units excluded according to criteria of viability arediscarded, and cannot proceed through further steps of processing fortransplantable cord blood units. Thawed cord blood units meeting thecriteria of viability can be assayed for the percent CD133+ and percentCD34+ cells in the thawed unit, and that data recorded for laterreference.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject, at least 1.5×10⁷ pre-cryopreservedtotal nucleated cells per kilogram subject weight and at least 8×10⁶total pre-cryopreserved CD34+ cells.

Thawed cord blood units deemed suitable, by viability, for furtherprocessing for ex-vivo expansion and transplantation are then separatedinto two cord blood fractions by selection according to stem cell markerCD133+. The CD133+/CD34+ cell fraction of the cord blood unit representsa hematopoietic stem and progenitor cell population with potential toproliferate, differentiate and reconstitute all blood cell lineages,while the unselected, CD133−/CD34− “negative” cell fraction represents afraction rich in mature immune cells such as B-cells. T-cells andnatural killer (NK) cells.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject and at least 1.8×10⁷pre-cryopreserved total nucleated cells.

Stem cells can be identified and enriched using stem cell markers suchas CD34+, CD34+/CD38−, CD133+, CD34+/Lin−, and other stem cell markersknown in the art. Identification and separation of the CD133+/CD34+fraction of the thawed cord blood cells can be performed by selectionusing FACS, immunomagnetic separation or nucleic acid methods such asPCR, such as the methods of isolating or enriching for cord blood andadult stem cells described in Stem Cell Biology Daniel R. Marshak(Editor) Richard L. Gardner (Editor), Publisher: Cold Spring HarborLaboratory Press, (2001) and Hematopoietic Stem Cell Transplantation.Anthony D. Ho (Editor) Richard Champlin (Editor), Publisher: MarcelDekker (2000).

In a particular embodiment, the thawed cord blood unit selected suitablefor ex-vivo expansion and transplantation is separated into aCD133+/CD34+ (positive) fraction and a CD133−/CD34− (negative) fractionusing anti-CD133-conjugated immunomagnetic beads (CliniMacs, MilentyiBiotec, Germany).

In specific embodiments, separation of the CD133+ fraction is affectedas follows: 1.2 ml of the Miltenyi CliniMACS CD133 reagent is added tothe cord blood cell suspension and incubated for 5 minutes at roomtemperature. Buffer is added and the suspension centrifuged for 10minutes at 450×g at room temperature. The supernatant is removed and thecell pellet is re-suspend in PBS buffer containing 0.4% w/v sodiumcitrate and 1% v/v HSA, and the cell suspension is transferred into atransfer bag. The CliniMACS system (Miltenyi Biotec, Germany) isassembled as per the manufacturer's instructions, and the selectedCD133+ cells are collected into a “collection” transfer bag while theCD133− unselected, non-binding cells are collected into the negativefraction bag. The cells are transferred aseptically from the“collection” bag into a 175 ml centrifuge tube. The selected cellsuspension is centrifuged at 450×g for 10 minutes at room temperature,the supernatant removed and the cells transferred into a sterile tubefor safety testing. The cell pellet is re-suspended and the exact volumemeasured and recorded.

A sample of the cell suspension is removed and the cell viability andthe viable cell concentration are measured. The total number of viablecells and the cell yield obtained from the column are calculated. Thepercentage of purified CD133+ and CD34+ cells is measured. The purity isdefined as the percent of CD133+ cells within the selected cellpopulation.

Once separated, the CD133+/CD34+ positive fraction can be expandedex-vivo, but must first be screened for suitability for furtherprocessing, Thus, in some embodiments, the method for selecting a thawedumbilical cord blood unit for ex-vivo expansion and transplantation intoa subject further comprises:

-   -   (a) separating said selected thawed umbilical cord blood unit        suitable for ex-vivo expansion transplantation into said subject        into (i) a first, selected blood cell fraction comprising        CD133+/CD34+ selected cells and (ii) a second, unselected blood        cell fraction comprising CD133/CD34 negative cells, and    -   (b) determining in said first, selected CD133+/CD34+ fraction        the following post-separation parameters:    -   (i) about 20×10⁵ to about 75×10⁵ total cells;    -   (ii) about 70% to about 85% viability;    -   (iii) about 70% to about 85% CD133+ cells;    -   (iv) about 70% to about 85% CD34+ cells;    -   (v) about 0.02 to about 1% yield post CD133+/CD34+ selection    -   (vi) about 15 to about 50 CFU/1000 cells, and    -   (vii) less than 3 Eu/ml endotoxin, and    -   (c) selecting or excluding said first, selected CD133+/CD34+        fraction according to said parameters.    -   thereby selecting a CD133+/CD34+ cord blood fraction suitable        for ex-vivo expansion and transplantation into said subject.

In particular embodiments, the CD133+/CD34+ cord blood fraction selectedfor ex-vivo expansion has total post-separation cell count of about20×10⁵ to about 75×10⁵ cells, about 25×10⁵ to about 65×10⁵ cells, about30×10⁵ to about 60×10⁵ cells, about 36×10⁵ to about 55×10⁵ cells andabout 40×10⁵ to about 50×10⁵ cells. In one embodiment, the CD133+/CD34+cord blood fraction selected for ex-vivo expansion has a totalpost-separation cell count of at least 20×10⁶, at least 30×10⁶, at least35×10⁶, at least 40×10⁶, at least 45×10⁶, at least 50×10⁶, at least55×10⁶, at least 60×10⁶ and at least 70×10⁶ total cells. In a furtherembodiment, the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion has a total post-separation cell count of at least 25×10⁶total cells. In a further embodiment, the CD133+/CD34+ cord bloodfraction selected for ex-vivo expansion has a total post-separation cellcount of at least 20×10⁶ total cells. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atotal post-separation cell count of at least 45×10⁶ total cells.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject and have at least 1.8×10⁹pre-cryopreserved total nucleated cells.

In some embodiments, cords blood units selected for ex-vivo expansionare pre-cryopreservation HLA-matched at at least 4 out of 6 HLA class I(HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, highresolution) loci with said subject and have at least 2.5×10⁹pre-cryopreserved total nucleated cells.

In some embodiments, CD133+/CD34+ cord blood fractions having about 70%to about 85% viability are selected. In some embodiments. CD133+/CD34+cord blood fractions having about 71%, about 73%, about 75%, about 78%,about 80%, about 82%, about 83%, about 84% to about 85% viability areselected. In a further embodiment, the CD133+/CD34+ cord blood fractionselected for ex-vivo expansion has at least 70% viable cells. In afurther embodiment, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 77% viable cells. In a furtherembodiment, the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion has at least 85% viable cells.

The CD133+, CD34+ and CD133+/CD34+ cell content of the separated cordblood fraction is critical to the successful engraftment of thetransplanted expanded cord blood cells, and is thus a central criterionfor proceeding to ex-vivo expansion. Thus, in particular embodiments,the CD133+/CD34+ cord blood fraction selected for ex-vivo expansion hastotal post-separation cell count of about 70% to about 85% CD133+ cells,about 73% to about 82% CD133+ cells, about 75% to 80% CD133+ cells andabout 76-79% CD133+ cells. In one embodiment, the CD133+/CD34+ cordblood fraction selected for ex-vivo expansion has a totalpost-separation CD133+ cell count of at least 70%, at least 74%, atleast 78%, at least 80%, at least 83% or at least 85% CD133+ cells. In afurther embodiment, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation CD133+ cell count of atleast 80% CD133+ cells post-separation for CD133+. In a furtherembodiment, the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion has a total post-separation CD133+ cell count of at least 70%CD133+ cells post-separation for CD133+. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atotal post-separation CD133+ cell count of at least 75% CD133+ cellspost-separation for CD133+.

Thus, in particular embodiments, the CD133+/CD34+ cord blood fractionselected for ex-vivo expansion has total post-separation cell count ofabout 70% to about 85% CD133+ cells, about 73% to about 82% CD133+cells, about 75% to 80% CD133+ cells and about 76-79% CD34+ cells. Inone embodiment, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation CD34+ cell count of atleast 70%, at least 74%, at least 78%, at least 80%, at least 83% or atleast 85% CD34+ cells. In a further embodiment, the CD133+/CD34+ cordblood fraction selected for ex-vivo expansion has a totalpost-separation CD34+ cell count of at least 82% CD34+ cellspost-separation for CD133+. In a further embodiment, the CD133+/CD34+cord blood fraction selected for ex-vivo expansion has a totalpost-separation CD34+ cell count of at least 70% CD133+ cellspost-separation for CD133+. In a further embodiment, the CD133+/CD34+cord blood fraction selected for ex-vivo expansion has a totalpost-separation CD34+ cell count of at least 78% CD133+ cellspost-separation for CD133+.

In particular embodiments, the post-selection yield of the CD133+/CD34+cord blood fraction selected for ex-vivo expansion is about 0.02% toabout 1.0%, about 0.05% to about 0.8%, about 0.08% to about 0.7%, about0.1% to about 0.55%, about 0.25% to about 0.45% and about 0.3-0.4%following separation for CD133+. In one embodiment, the post-selectionyield of the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion is at least 0.01%, at least 0.05%, at least 0.1%, at least0.3%, at least 0.5%, at least 0.65%, at least 0.75%, at least 0.9% or atleast 1.0% post-selection for CD133+. In a further embodiment, thepost-selection yield of the CD133+/CD34+ cord blood fraction selectedfor ex-vivo expansion is at least 0.02% post-separation. In a furtherembodiment, the post-selection yield of the CD133+/CD34+ cord bloodfraction selected for ex-vivo expansion is at least 0.3%post-separation. In a further embodiment, the post-selection yield ofthe CD133+/CD34+ cord blood fraction selected for ex-vivo expansion isat least 0.5% post-separation. In a further embodiment, thepost-selection yield of the CD133+/CD34+ cord blood fraction selectedfor ex-vivo expansion is at least 0.8% post-separation.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has about 15 to about 75 CFU per 1000 cells, about 20to about 60 CFU per 1000 cells, about 25 to about 50 CFU per 1000 cells,about 30 to about 40 CFU per 1000 cells post-separation. In oneembodiment, the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion has at least 15 CFU per 1000 cells, at least 22 CFU per 1000cells, at least 25 CFU per 1000 cells, at least 30 CFU per 1000 cells,at least 36 CFU per 1000 cells, at least 40 CFU per 1000 cells, at least45 CFU per 1000 cells, at least 52 CFU per 1000 cells, at least 60 CFUper 1000 cells, at least 68 CFU per 1000 cells or at least 75 CFU per1000 cells post-selection for CD133+. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atleast 15 CFU per 1000 cells following selection for CD133+. In a furtherembodiment, the CD133+/CD34+ cord blood fraction selected for ex-vivoexpansion has at least 25 CFU per 1000 cells following selection forCD133+. In a further embodiment, the CD133+/CD34+ cord blood fractionselected for ex-vivo expansion has at least 40 CFU per 1000 cellsfollowing selection for CD133+. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atleast 45 CFU per 1000 cells following selection for CD133+.

Examples of exemplary criteria for selection of thawed cord blood unitssuitable for ex-vivo expansion and transplantation into a subject arepresented in Tables V-VIII hereinbelow.

TABLE V Cord Blood Selection criteria for Expansion Parameter ValueTotal cells At least 35 × 10⁵ Viability At least 85% CD133+ cellfraction At least 77% CD34+ cell fraction At least 83% CFU per 1000cells At least 23 Yield post-selection At least 0.5% Endotoxin No morethan 3 Eu/ml

TABLE VI Cord Blood Selection criteria for Expansion Parameter ValueTotal cells At least 20 × 10⁵ Viability At least 70% CD133+ cellfraction At least 70% CD34+ cell fraction At least 70% CFU per 1000cells At least 15 Yield post-selection At least 0.02% Endotoxin No morethan 3 Eu/ml

TABLE VII Cord Blood Selection criteria for Expansion Parameter ValueTotal cells At least 45 × 10⁵ Viability At least 85% CD133+ cellfraction At least 72% CD34+ cell fraction At least 83% CFU per 1000cells At least 18 Yield post-selection At least 0.5% Endotoxin No morethan 3 Eu/ml

TABLE VIII Cord Blood Selection criteria for Expansion Parameter ValueTotal cells At least 55 × 10⁵ Viability At least 76% CD133+ cellfraction At least 85% CD34+ cell fraction At least 76% CFU per 1000cells At least 35 Yield post-selection At least 0.09% Endotoxin No morethan 3 Eu/ml

Sterility and safety of the thawed, CD133+/CD34+ cord blood unit forex-vivo expansion and transplantation is assured by monitoring, interalia, the endotoxin content and presence of bacterial, fungal, viral andmycoplasma contamination. In some embodiments, the CD133+/CD34+ cordblood fraction selected for ex-vivo expansion has an endotoxin contentof no more than 3 Eu/ml at any time during the expansion (cellculturing) process. In some embodiments, the CD133+/CD34+ cord bloodfraction selected for ex-vivo expansion is free of bacterial, yeast,mold and mycoplasm following selection for CD133+ and on any day tested.In some embodiments, endotoxin content is monitored on days 0 and 7 ofthe ex-vivo expansion. In some embodiments, bacteria, yeast, mold andmycoplasma are monitored on days 0, 7 and 14 of the ex-vivo expansion.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 15 CFU per 1000 cells and apost-selection yield of at least 0.02%. In other embodiments, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atleast 15 CFU per 1000 cells, a post-selection yield of at least 0.02%and a total post-separation CD133+ cell count of at least 75% CD133+cells post-separation for CD133+. In other embodiments, the CD133+/CD34+cord blood fraction selected for ex-vivo expansion has at least 35 CFUper 1000 cells and a post-selection yield of at least 0.1%.

In other embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 15 CFU per 1000 cells and a totalpost-separation CD133+ cell count of at least 70% CD133+ cellspost-separation for CD133+.

The unselected negative, CD133− fraction comprises the repertory ofimmune cells, including, but not limited to T lymphocytes. B lymphocytesand Natural Killer (NK) cells. Following selection of the thawed cordblood cells for CD133+ cells, unselected negative, CD133− fraction isprepared for cryopreservation by washing and suspension incryopreservation solution. Many cryopreservation solutions arecommercially available. In some embodiments, the cryopreservationsolution is CryoStor®CS10 (BioLife Solutions. Inc). Prior tocryopreservation, the unselected, CD133/CD34 negative cord blood cellfraction is also monitored for parameters including viability, CD3+ cell(T-cell) content. CD133+/CD34+ content and sterility. Thus, in someembodiments, the present invention provides a method of selectingunselected umbilical cord blood cell fractions comprising CD133/CD34negative cells for transplantation into a subject, comprising:

-   -   (a) determining in said uncultured umbilical cord blood fraction        following selection for CD133/CD34 negative cells the following        parameters:        -   (i) about 4×10⁸ to about 15×10⁸ total viable cells;        -   (ii) about 70-85% viability of the cells;        -   (iii) about 2.4×10⁷ to about 8×10⁷ CD3+ cells;        -   to (iv) no bacterial, yeast or mold growth, and    -   (b) selecting or excluding said unselected umbilical cord blood        cell fraction according to said parameters,    -   thereby selecting an unselected umbilical cord blood cell        fraction comprising CD133/CD34 negative cells suitable for        transplantation into the subject.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation cell count of at least20×10⁶ total cells and at least 70% viable post-separation cells.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation cell count of at least20×10⁶ total cells, and at least 80% CD133+ cells.

In other embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 15 CFU per 1000 cells, a post-selectionyield of at least 0.02% and a total post-separation CD34+ cell count ofat least 70% CD34+ cells post-separation for CD133+.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving about 70% to about 85% viability are selected fortransplantation. In some embodiments, the CD133/CD34 negative cord bloodfractions having about 71%, about 73%, about 75%, about 78%, about 80%,about 82%, about 83%, about 84% to about 85% viability are selected. Ina further embodiment, the CD133/CD34 negative cord blood fractionselected for transplantation has at least 70% viable cells.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation cell count of at least20×10⁶ total cells, and at least 70% CD133+ cells.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has a total post-separation cell count of at least20×10⁶ total cells, and at least 70% CD34+ cells.

In other embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 15 CFU per 1000 cells, a post-selectionyield of at least 0.02% and a total post-separation CD34+ cell count ofat least 85% CD133+ cells post-separation for CD133+.

In other embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 15 CFU per 1000 cells, a post-selectionyield of at least 0.02% and a total post-separation CD133+ cell count ofat least 70% CD133+ cells post-separation for CD133+.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 70% viability and at least 70% CD34+cells.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving about 4×10⁸ to about 15×10⁸ total viable cells are selected fortransplantation.

In some embodiments, the number of total viable cells in the CD133/CD34negative fraction selected for transplantation is about 6×10⁸ to about13×10⁸ cells, about 7.5×10⁸ to about 11×10⁸ viable cells or about 9×10⁸to about 10×10⁸ viable cells. In some embodiments, the number of totalviable cells in the CD133/CD34 negative fraction selected fortransplantation is at least 4×10⁸, at least 6×10⁸, at least 7.5×10⁸, atleast 9×10⁸, at least 10×10⁸, at least 12×10⁸, at least 14×10⁸ or atleast 15×10⁸ total viable cells. In a particular embodiment, the numberof total viable cells in the CD133/CD34 negative fraction selected fortransplantation is at least 4×10⁸ total viable cells.

In some embodiments, the CD133+/CD34+ cord blood fraction selected forex-vivo expansion has at least 70% viable cells and a post-selectionyield of at least 0.02% CD133+/CD34+ cells. In some embodiments, theCD133+/CD34+ cord blood fraction selected for ex-vivo expansion has atleast 80% CD34+ cells, 85% viable cells and a post-selection yield of atleast 0.1% CD133+/CD34+ cells. In some embodiments, the CD133+/CD34+cord blood fraction selected for ex-vivo expansion has at least 70%CD34+ cells, 80% viable cells and a post-selection yield of at least0.02% CD133+/CD34+ cells.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving about 2.4×10⁷ to about 8×10⁷ CD3+ cells are selected fortransplantation. In some embodiments, unselected CD133/CD34 negativecord blood fractions having about 2.4×10⁷ to about 8×10⁷ CD3+ cells,about 3.5×10⁷ to about 7×10⁷ CD3+ cells, about 4.3×10⁷ to about 6.2×10⁷CD3+ cells or about 4.8×10⁷ to about 5×10⁷ CD3+ cells are selected fortransplantation. In other embodiments, unselected CD133/CD34 to negativecord blood fractions having at least 2.4×10⁷ CD3+ cells, at least3.0×10⁷ CD3+ cells, at least 3.5×10⁷ CD3+ cells, at least 4.0×10⁷ CD3+cells, at least 4.5×10⁷ CD3+ cells, at least 5×10⁷ CD3+ cells, at least6×10⁷ CD3+ cells, at least 7×10⁷ CD3+ cells or at least 8×10⁷ CD3+ cellsare selected for transplantation. In some embodiments, the unselectedCD133/CD34 negative cord blood fractions selected for transplantationhave at least 3.0×10⁷ CD3+ cells. In some embodiments, the unselectedCD133/CD34 negative cord blood fractions selected for transplantationhave at least 2.4×10⁷ CD3+ cells. In some embodiments, the unselectedCD133/CD34 negative cord blood fractions selected for transplantationhave at least 5×10⁷ CD3+ cells.

In other embodiments, unselected CD133/CD34 negative cord bloodfractions having at least 5×10⁷ CD3+ cells and 6×10⁸ total viable cellsare selected for transplantation. In some embodiments, unselectedCD133/CD34 negative cord blood fractions having at least 2.4×10⁷ CD3+cells and 4×10⁸ total viable cells are selected for transplantation. Instill other embodiments, unselected CD133/CD34 negative cord bloodfractions having at least 6×10⁷ CD3+ cells and 4×10⁸ total viable cellsare selected for transplantation.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving about 0.01% to about 0.5% CD133+/CD34+ cells are selected fortransplantation. In some embodiments, the CD133/CD34 negative cord bloodfractions having no more than about 0.03%, about 0.05%, about 0.08%,about 0.1%, about 0.25%, about 3%, about 4% or about 5% CD133+/CD34+cells are selected. In a further embodiment, the CD133/CD34 negativecord blood fraction selected for transplantation has no greater than0.01% CD133+/CD34+ cells. In a further embodiment, the CD133/CD34negative cord blood fraction selected for transplantation has no greaterthan 0.05% CD133+/CD34+ cells. In a further embodiment, the CD133/CD34negative cord blood fraction selected for transplantation has no greaterthan 0.1% CD133+/CD34+ cells. In a further embodiment, the CD133/CD34negative cord blood fraction selected for transplantation has no greaterthan 0.25% CD133+/CD34+ cells. In a further embodiment, the CD133/CD34negative cord blood fraction selected for transplantation has no greaterthan 0.5% CD133+/CD34+ cells.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving at least 4×10⁸ total viable cells and 85% viability are selectedfor to transplantation. In some embodiments, unselected CD133/CD34negative cord blood fractions having at least 70% viability and 2.4×10⁷CD3+ cells are selected for transplantation.

Cryopreserved CD133/CD34 negative cord blood fractions selected suitablefor transplantation can be stored and maintained frozen until needed fortransplantation. In some embodiments, the CD133/CD34 negative cord bloodfractions selected suitable for transplantation are stored in liquidnitrogen.

In some embodiments, unselected CD133/CD34 negative cord blood fractionshaving at least 4×10⁸ total viable cells and 70% viability are selectedfor transplantation. In some embodiments, unselected CD133/CD34 negativecord blood fractions having at least 70% viability and 2.4×10⁷ CD3+cells are selected for transplantation. In some embodiments, unselectedCD133/CD34 negative cord blood fractions having at least 4×10⁸ totalviable cells, at least 2.4×10⁷ CD3+ cells and 70% viability are selectedfor transplantation. In some embodiments, unselected CD133/CD34 negativecord blood fractions having at least 6×10⁸ total viable cells, at least2.7×10⁷ CD3+ cells and at least 75% viability are selected fortransplantation. In some embodiments, unselected CD133/CD34 negativecord blood fractions having at least 4×10⁸ total viable cells and atleast 80% viability are selected for transplantation. Separation andhandling of the unselected CD133/CD34 negative cord blood fractions cancomprise monitoring the cultured units for safety and contamination.

Thus, according to specific embodiments, cord blood fractions or unitshaving greater than 3.0 Eu/ml endotoxin, and/or bacterial, yeast or moldgrowth are excluded from cryopreservation or, if detected at any pointduring the preparation process, further processing is terminated and thecontaminated units discarded.

Culture (Ex-vivo expansion) of CD133+/CD34+ Selected Fractions

Any of the methods for selection of cord blood units for ex-vivoexpansion described hereinabove and each of their embodiments takenalone or in various combinations may be used for affecting the methodsfor culturing (Ex-vivo expansion) of CD133+/CD34+ selected fractions asis described in this section and the sections that follow.

Nicotinamide (NAM) is a water-soluble derivative of vitamin B, whose tophysiological active forms are nicotinamide adenine dinucleotide(NAD+/NADH) and nicotinamide adenine dinucleotide phosphate(NADP+/NADPH). The physiological active forms of NAM serve as coenzymein a variety of important metabolic reactions. Nicotinamide is furtherknown to inhibit the enzymatic activity of CD38, to thereby affect thecADPR signal transduction pathway.

The methods for ex-vivo expanding the CD133+CD34+ fraction selectedsuitable for expansion and transplantation of the present invention canbe performed by providing the cells either with nicotinamide itself, orwith a nicotinamide analog, a nicotinamide or a nicotinamide analogderivative or a nicotinamide or a nicotinamide analog metabolite.

As used herein, the phrase “nicotinamide analog” refers to any moleculethat is known to act similarly to nicotinamide. Representative examplesof nicotinamide analogs include, without limitation, benzamide,nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid andα-amino-3-indolepropionic acid. The phrase “a nicotinamide or anicotinamide analog derivative” refers to any structural derivative ofnicotinamide itself or of an analog of nicotinamide. Examples of suchderivatives include, without limitation, substituted benzamides,substituted nicotinamides and nicotinethioamides and N-substitutednicotinamides and nicotinthioamides. The phrase “a nicotinamide or anicotinamide analog metabolite” refers to products that are derived fromnicotinamide or from analogs thereof such as, for example, NAD, NADH andNADPH. In particular embodiments, the CD133+CD34+ fraction selectedsuitable for expansion and transplantation is cultured with nicotinamidefor ex-vivo expansion.

Final concentrations of the nicotinamide or of the analogs, derivativesor metabolites thereof are preferably, depending on the specificapplication, in the millimolar ranges. For example, within about 1 mM toabout 10 mM, within about 2.5 mM to about 8 mM or within about 2.5 mM toabout 5 mM.

According to further features in preferred embodiments of the inventiondescribed herein, conditions for ex vivo cell proliferation comprisesproviding the cells with scrum, nutrients and cytokines.

The cytokines can be early acting cytokines. In some embodiments, theearly acting cytokines are selected from the group comprising stem cellfactor, FLT3 ligand, interleukin-1, interleukin-2, interleukin-3,interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor-αand thrombopoietin. In specific embodiments, the cytokines are acombination of cytokines including each of stem cell factor,thrombopoietin. FLt3 ligand, and IL-6.

According to the methods of the present invention, the CD133+CD34+ cordblood fraction selected suitable for expansion and transplantation isex-vivo expanded by culturing the first, selected blood cell fractioncomprising CD133+CD34+ selected cells ex vivo under conditions allowingfor cell proliferation, said conditions which comprise providingnutrients, serum and a combination of cytokines including each of stemcell factor, thrombopoietin. FLt3 ligand, and IL-6 and, in the sameculture medium, nicotinamide in an amount between 1.0 to 10.0 mM.

Thus, according the present invention, there is provided a method forpreparing an umbilical cord blood unit for transplantation into asubject, the method comprising:

-   -   (a) separating a single, thawed umbilical cord blood unit        suitable for transplantation into said subject into (i) a first,        selected blood cell fraction comprising CD133+/CD34+ selected        cells and (ii) a second, unselected blood cell fraction        comprising CD133/CD34 negative cells; and    -   (b) ex vivo culturing said first blood cell fraction comprising        CD133+/CD34+ selected cells under conditions allowing for cell        proliferation, said conditions comprising providing nutrients,        serum and cytokines including each of stem cell factor,        thrombopoietin. FLt3 ligand and TL-6 and nicotinamide in an        amount between 1.0 mM to 10 mM.

In some embodiments, the single umbilical cord blood unit suitable fortransplantation into a subject is a thawed cord blood unit selectedsuitable for ex-vivo expansion and transplantation into a subjectaccording to the methods described herein for selecting cryopreservedumbilical cord blood for expansion and transplantation into a subject.For example, thawed single umbilical cord blood units suitable fortransplantation into a subject can be characterized by (i) about 8×10⁶to about 15×10⁶ total CD34+ cells, (ii) HLA-matched at at least 4 out of6 HLA class I (HLA-A and HLA-B, low resolution) and HLA class II(HLA-DRB1, high resolution) loci with said subject, (iii) about 1.8×10⁹to about 3.0×10⁹ pre-cryopreserved total nucleated cells and (iv) about1.5×10⁷ to about 3.0×10⁹ pre-cryopreserved total nucleated cells perkilogram subject weight.

In some embodiments, the first, selected CD133+/CD34+ blood cellfraction (a)(i) is a CD133+/CD34+ cord blood fraction selected suitablefor ex-vivo expansion and transplantation into said subject according tothe methods described herein for selecting a CD133+/CD34+ cord bloodfraction suitable for ex-vivo expansion and transplantation into asubject. For example, the CD133+/CD34+ cord blood fraction suitable forex-vivo expansion and transplantation into a subject can becharacterized by (i) about 8×10⁶ to about 15×10⁶ total CD34+ cells; (ii)HLA-matching at at least 4 out of 6 HLA class I (HLA-A and HLA-B, lowresolution) and HLA class II (HLA-DRB1, high resolution) loci with saidsubject; (iii) about 1.8×10⁹ to about 3.0×10⁹ pre-cryopreserved totalnucleated cells; and (iv) about 1.5×10⁷ to about 3.0×10⁷pre-cryopreserved total nucleated cells per kilogram subject weight.

In further embodiments of the method of the present invention, cordblood fractions or units having greater than 3.0 Eu/ml endotoxin, and/orbacterial, yeast or mold growth at day 0, day 7 or at day 14 of saidex-vivo expansion are excluded from culturing (ex-vivo expansion).

In further embodiments, the serum in the culture medium for expansion ofthe CD133+/CD34+ cord blood fraction suitable for ex-vivo expansion andtransplantation into a subject comprises 10% FBS and 50 ng each of steincell factor, thrombopoietin, FLt3 ligand and IL-6. In specificembodiments, the culture medium for expansion of the CD133+/CD34+ cordblood fraction comprises nicotinamide at 2.5 mM. In further embodiments,culturing the cells under conditions allowing for cell proliferationcomprises providing nutrients, 10% fetal bovine serum (FBS), andcytokines including 50 ng each of stem cell factor, thrombopoietin, FLt3ligand and IL-6 and nicotinamide at 2.5 mM. In some embodiments, thenicotinamide is provided at 5.0 mM.

In some embodiments, the CD133+/CD34+ cord blood fraction suitable forex-vivo expansion and transplantation into a subject is cultured forex-vivo expansion by culturing under the conditions described herein for18-25 days. In specific embodiments, the CD133+/CD34+ cord bloodfraction is cultured for 18, for 19, for 20, for 21, for 22, for 23, 24or for 25 days. In some embodiments, the CD133+/CD34+ cord bloodfraction is cultured for ex-vivo expansion for 21 days.

As mentioned herein, culturing the CD133+/CD34+ cord blood fraction forex-vivo expansion can comprise monitoring the cultured units for safelyand contamination.

Thus, according to specific embodiments, cord blood fractions or unitshaving greater than 3.0 Eu/ml endotoxin, and/or bacterial, yeast or moldgrowth at day 7 or at day 14 of said ex-vivo expansion are excluded fromculturing (ex-vivo expansion) or, if detected during the culturingprocess, culturing (expansion) is terminated and the contaminated unitsdiscarded.

Selection of Ex-Vivo Expanded Cord Blood Units for Transplantation

At completion of the culturing (ex-vivo expansion) of the CD133+/CD34+cord blood fraction selected suitable for ex-vivo expansion, the ex-vivocultured first selected CD133+/CD34+ can be harvested for storage.

Any of the methods for selection of cord blood units for ex-vivoexpansion described hereinabove, and for expansion (ex-vivo culturing)of cord blood units described hereinabove, and each of their embodimentstaken alone or in various combinations may be used for affecting themethods for selecting cultured (ex-vivo expanded) CD133+/CD34+ selectedfractions as is described in this section and the sections that follow.

Prior to storage, the ex-vivo expanded CD133+CD34+ cord blood fractionis characterized for suitability for cryopreservation as an expandedCD133+CD34+ cord blood fraction for transplantation into a subject.Thus, according to the present invention, there is provided a method ofselecting ex-vivo cultured umbilical cord blood cell fractionscomprising CD133+CD34+ selected cord blood cells for transplantationinto a subject, the method comprising (a) determining in said ex-vivocultured umbilical cord blood fraction following ex-vivo expansion thefollowing parameters:

-   -   (i) about 8×10⁸ to about 15×10⁸ total viable cells;    -   (ii) about 70%-85% viability of the cells;    -   (iii) about 7-15% CD34+ cells;    -   (iv) about 5.6×10⁷ to about 5×10⁸ total CD34+ cells;    -   (v) about 2.4×10⁷ to about 2×10⁸ total CD133+ cells;    -   (vi) about 8×10⁵ to about 25×10⁵ CD133+/CD38− cells;    -   (vii) about 8×10⁷ to about 15×10⁹ total CD14+ cells.    -   (viii) about 2×10⁸ to about 2×10⁹ total CD15+ cells,    -   (ix) about 8×10⁷ to about 8×10⁹ total CD11b+ cells.    -   (x) about 3.2×10⁷ to about 3×10⁸ total CD1(a+c)+ cells.    -   (xi) No cultured mycoplasma or bacterial, yeast or mold growth,        and    -   (b) selecting or excluding said ex-vivo cultured umbilical cord        blood fraction according to said parameters,    -   thereby selecting an ex-vivo cultured CD133+/CD34+ umbilical        cord blood fraction suitable for transplantation into the        subject.

In some embodiments, ex-vivo expanded CD133+/CD34+ cord blood fractionshaving total of about 8×10⁸ to about 15×10⁸ total viable cells areselected. In some embodiments, ex-vivo expanded CD133+/CD34+ cord bloodfractions having total of about 9×10⁸ to about 13×10⁸ total viablecells, about 10×10⁸ to about 12×10⁸ or about 10.5×10⁸ to about 11×10⁸total viable cells are selected for transplantation. In one embodiment,the ex-vivo expanded CD133+/CD34+ cord blood fraction selected fortransplantation has at least 8×10⁸, at least 10×10⁸, at least 11×10⁸, atleast 12×10⁸, at least 13×10⁸, at least 14×10⁸ and at least 15×10⁸ totalviable cells. In a particular embodiment, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has atleast 8×10⁸ total viable cells. In a particular embodiment, the ex-vivoexpanded CD133+/CD34+ cord blood fraction selected for transplantationhas at least 11.5×10⁸ total viable cells. In a particular embodiment,the ex-vivo expanded CD133+/CD34+ cord blood fraction selected fortransplantation has at least 15×10⁸ total viable cells.

In some embodiments, ex-vivo expanded CD133+/CD34+ cord blood fractionshaving about 70% to about 85% viability are selected. In someembodiments, ex-vivo expanded CD133+/CD34+ cord blood fractions havingabout 71%, about 73%, about 75%, about 78%, about 80%, about 82%, about83%, about 84% to about 85% viability are selected for transplantation.In some embodiments, ex-vivo expanded CD133+/CD34+ cord blood fractionshaving at least 71%, at least 73%, at least 75%, at least 78%, at least80%, at least 82%, at least 83%, at least 84% or at least 85% viabilityare selected. In a further embodiment, the ex-vivo expanded CD133+/CD34+cord blood fraction selected for transplantation has at least 70% viablecells. In a further embodiment, the ex-vivo expanded CD133+/CD34+ cordblood fraction selected for transplantation has at least 78% viablecells. In a further embodiment, the ex-vivo expanded CD133+/CD34+ cordblood fraction selected for transplantation has at least 83% viablecells.

The CD133+, CD34+ and CD133+/CD34+, CD133+/CD38−, CD14+, CD15+, CD11b+and CD1(a and c)+ cell content of the ex-vivo expanded CD133+/CD34+ cordblood fraction is critical to the successful engraftment of thetransplanted expanded cord blood cells, and is thus a central criterionfor selecting proceeding to ex-vivo expansion.

Thus, in a particular embodiment, an ex-vivo expanded CD133+/CD34+ cordblood fraction selected for transplantation has a total post-expansion(post-culturing) portion of CD34+ cells of at least 7%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, at least 13%, atleast 14% or at least 15% CD34+ cells. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for transplantation has atotal post-expansion (post-culturing) portion of CD34+ cells of at least7%. In a further embodiment, the CD133+/CD34+ cord blood fractionselected for transplantation has a total post-expansion (post-culturing)portion of CD34+ cells of at least 7.8%. In a further embodiment, theCD133+/CD34+ cord blood fraction selected for transplantation has atotal post-expansion (post-culturing) portion of CD34+ cells of at least9%. In a further embodiment, the CD133+/CD34+ cord blood fractionselected for transplantation has a total post-expansion (post-culturing)portion of CD34+ cells of at least 13%.

Thus, in particular embodiments, the ex-vivo expanded CD133+/CD34+ cordblood fraction selected for transplantation has total post-expansion(post-culturing) CD34+ cell count of about 5.6×10⁷ to about 5×10⁸ CD34+cells. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD34+ cell count of about 6×10⁷ to about 4×10⁸, about9×10⁷ to about 3×10⁸, about 1×10⁸ to about 2.5×10⁸, about 1.5×10⁸ toabout 2×10⁸ total CD34+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD34+ cell count of at least 5.6×10⁷, atleast 6×10⁷, at least 8×10⁷, at least 9×10⁷, at least 1×10⁸, at least2×10⁸, at least 3×10⁸, at least 4×10⁸ or at least 5×10⁸ total CD34+cells. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD34+ cell count of at least 2×10⁸ total CD34+ cells.In to some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD34+ cell count of at least 8×10⁷ total CD34+ cells.In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD34+ cell count of at least 5.6×10⁷ total CD34+ cells.

In particular embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD133+ cell count of about 2.4×10⁷ to about 2×10⁸CD133+ cells. In some embodiments, the ex-vivo expanded CD133+/CD34+cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD133+ cell count of about 3×10⁷ toabout 1×10⁸, about 5×10⁷ to about 8×10⁷, about 6×10⁷ to about 7×10⁷total CD133+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD133+ cell count of at least 2.4×10⁷,at least 3.5×10⁷, at least 5×10⁷, at least 7×10⁷, at least 8×10⁷, atleast 1×10⁸, at least 1.5×10⁸ or at least 2×10⁸ total CD133+ cells. Insome embodiments, the ex-vivo expanded CD133+/CD34+ cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD133+ cell count of at least 2.4×10⁷ total CD133+ cells. In someembodiments, the ex-vivo expanded CD133+/CD34+ cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD133+ cell count of at least 4×10⁷ total CD133+ cells. In someembodiments, the ex-vivo expanded CD133+/CD34+ cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD133+ cell count of at least 7.5×10⁷ total CD133+ cells. In someembodiments, the ex-vivo expanded CD133+/CD34+ cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD133+ cell count of at least 1.2×10⁸ total CD133+ cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD133+/CD38− cell count of about 8×10⁵ to about 25×10⁵CD133+/CD38− cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD133+/CD38− cell count of about 10×10⁵to about 22×10⁵, about 13×10⁵ to about 20×10⁵ or about 15×10⁵ to about17×10⁵ total CD133+/CD38− cells. In some embodiments, the ex-vivoexpanded to CD133+/CD34+ cord blood fraction selected fortransplantation has total post-expansion (post-culturing) CD133+/CD38−cell count of at least 8×10⁵, at least 10×10⁵, at least 11.5×10⁵, atleast 13×10⁵, at least 15×10⁵, at least 18×10⁵, at least 20.0×10⁵, atleast 22×10⁵ or at least 25×10⁵ total CD133+ cells. In some embodiments,the ex-vivo expanded CD133+/CD34+ cord blood fraction selected fortransplantation has total post-expansion (post-culturing) CD133+/CD38−cell count of at least 13×10⁵. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD133+/CD38− cell count of at least8×10⁵. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD133+/CD38− cell count of at least 10×10⁵.

In yet further embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD14+ cell count of about 8×10⁷ to about 15×10⁸ CD14+cells. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD14+ cell count of about 10×10⁷ to about 10×10⁸, about12×10⁷ to about 8×10⁸, about 15×10⁷ to about 6×10⁸, about 2×10⁸ to about5×10⁸ or about 3×10⁸ to about 4×10⁸ CD14+ cells. In some embodiments,the ex-vivo expanded CD133+/CD34+ cord blood fraction selected fortransplantation has total post-expansion (post-culturing) CD14+ cellcount of at least 8×10⁷, at least 10×10⁷, at least 2×10⁸, at least4×10⁸, at least 6×10⁸, at least 8×10⁸, at least 10.0×10⁸, at least12×10⁸ or at least 15×10⁸ total C14+ cells. In some embodiments, theex-vivo expanded CD133+/CD34 cord blood fraction selected fortransplantation has total post-expansion (post-culturing) CD14+ cellcount of at least 8×10⁷. In some embodiments, the ex-vivo expandedCD133+/CD34 cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD14+ cell count of at least 2×10⁸. Insome embodiments, the ex-vivo expanded CD133+/CD34 cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD14+ cell count of at least 7.5×10⁸.

In yet further embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD15+ cell count of about 2×10⁸ to about 2×10⁹ CD15+cells. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD15+ cell count of about 2.5×10⁸ to about 1.5×10⁹,about 4×10⁸ to about 0.5×10⁹, about 6×10⁸ to about 9×10⁸ or about 7×10⁸to about 8×10⁸ CD15+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD15+ cell count of at least 2×10⁸, atleast 4×10⁸, at least 6×10⁸, at least 8×10⁸, at least 1×10⁹ or at least2×10⁹ total CD15+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34 cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD15+ cell count of at least 2×10⁸. Insome embodiments, the ex-vivo expanded CD133+/CD34 cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD15+ cell count of at least 6×10⁸. In some embodiments, the ex-vivoexpanded CD133+/CD34 cord blood fraction selected for transplantationhas total post-expansion (post-culturing) CD15+ cell count of at least8.5×10⁸. In some embodiments, the ex-vivo expanded CD133+/CD34 cordblood fraction selected for transplantation has total post-expansion(post-culturing) CD15+ cell count of at least 1.1×10⁹.

In yet further embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD11b+ cell count of about 8×10⁷ to about 8×10⁹ CD11b+cells. In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD11b+ cell count of about 1×10⁸ to about 5×10⁹, about4×10⁸ to about 2×10⁹, about 6×10⁸ to about 1×10⁸ or about 8×10⁸ to about9×10⁸ CD11b+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD11b+ cell count of at least 8×10⁷, atleast 1×10⁸, at least 4×10⁸, at least 6×10⁸, at least 8×10⁸, at least1×10⁹, at least 2×10⁹, at least 4×10⁹, at least 6×10⁹ or at least 8×10⁹total C11b+ cells. In some embodiments, the ex-vivo expanded CD133+/CD34cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD11b+ cell count of at least 8×10⁷. Insome embodiments, the ex-vivo expanded CD133+/CD34 cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD11b+ cell count of at least 10×10⁷. In some embodiments, the ex-vivoexpanded CD133+/CD34 cord blood fraction selected for transplantationhas total post-expansion (post-culturing) CD11b+ cell count of at least2×10⁸. In some embodiments, the ex-vivo expanded CD133+/CD34 cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD11b+ cell count of at least 5×10⁸. In someembodiments, the ex-vivo expanded CD133+/CD34 cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD11b+ cell count of at least 3×10⁹.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cord bloodfraction selected for transplantation has total post-expansion(post-culturing) CD1(a and c)+ cell count of about 3.2×10⁷ to about3×10⁸ CD15+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD1(a and c)+ cell count of about 4×10⁷to about 1×10⁸, about 6×10⁷ to about 9×10⁷ or about 7×10⁷ to about 8×10⁷CD1(a and c)+ cells. In some embodiments, the ex-vivo expandedCD133+/CD34+ cord blood fraction selected for transplantation has totalpost-expansion (post-culturing) CD1(a and c)+ cell count of at least3.2×10⁷, at least 4×10⁷, at least 6×10⁷, at least 8×10⁷, at least 1×10⁸or at least 3×10⁸ total CD1(a and c)+ cells. In some embodiments, theex-vivo expanded CD133+/CD34+ cord blood fraction selected fortransplantation has total post-expansion (post-culturing) CD1(a and c)+cell count of at least 3.2×10⁷. In some embodiments, the ex-vivoexpanded CD133+/CD34+ cord blood fraction selected for transplantationhas total post-expansion (post-culturing) CD1(a and c)+ cell count of atleast 5×10⁷. In some embodiments, the ex-vivo expanded CD133+/CD34+ cordblood fraction selected for transplantation has total post-expansion(post-culturing) CD1(a and c)+ cell count of at least 7.3×10⁷. In someembodiments, the ex-vivo expanded CD133+/CD34+ cord blood fractionselected for transplantation has total post-expansion (post-culturing)CD1(a and c)+ cell count of at least 2.2×10⁸.

Examples of exemplary post-expansion criteria for selection of cordblood units for ex-vivo transplantation into a subject are presented inTables IX-XII hereinbelow.

TABLE IX Selected, Expanded Cord Blood criteria for TransplantationParameter Value Total viable cells At least 12 × 10⁸ Viability At least76% CD34+ cell fraction At least 10% Total CD34+ cells At least 7 × 10⁷Total CD133+ cells At least 2.9 × 10⁷ Total CD133+/CD38− cells At least11 × 10⁵ Total CD14+ cells At least 8 × 10⁷ Total CD15+ cells At least 4× 10⁸ Total CD11b+ cells At least 1 × 10⁸ Total CD1(a and c)+ cells Atleast 8 × 10⁷

TABLE X Selected, Expanded Cord Blood criteria for TransplantationParameter Value Total viable cells At least 8 × 10⁸ Viability At least70% CD34+ cell fraction At least 7% Total CD34+ cells At least 5.6 × 10⁷Total CD133+ cells At least 2.4 × 10⁷ Total CD133+/CD38− cells At least8 × 10⁵ Total CD14+ cells At least 8 × 10⁷ Total CD15+ cells At least 2× 10⁸ Total CD11b+ cells At least 8 × 10⁷ Total CD1(a and c)+ cells Atleast 3.2 × 10⁷

TABLE XI Selected, Expanded Cord Blood criteria for TransplantationParameter Value Total viable cells At least 8 × 10⁸ Viability At least80% CD34+ cell fraction At least 11% Total CD34+ cells At least 1 × 10⁸Total CD133+ cells At least 4 × 10⁷ Total CD133+/CD38− cells At least1.6 × 10⁶ Total CD14+ cells At least 2 × 10⁸ Total CD15+ cells At least7 × 10⁸ Total CD11b+ cells At least 1.3 × 10⁸ Total CD1(a and c)+ cellsAt least 6 × 10⁷

TABLE XII Selected, Expanded Cord Blood criteria for TransplantationParameter Value Total viable cells At least 11 × 10⁸ Viability At least72% CD34+ cell fraction At least 11% Total CD34+ cells At least 6.7 ×10⁷ Total CD133+ cells At least 5.8 × 10⁷ Total CD133+/CD38− cells Atleast 9.3 × 10⁵ Total CD14+ cells At least 11 × 10⁷ Total CD15+ cells Atleast 6.9 × 10⁸ Total CD11b+ cells At least 21 × 10⁷ Total CD1(a and c)+cells At least 9.2 × 10⁷

As described hereinabove, parameters of safety and contamination aremonitored to ensure that no ex-vivo expanded CD133+/CD34+ cord bloodfractions having mycoplasma or bacterial, yeast or mold growth areselected for transplantation. Thus, in some embodiments, the ex-vivoexpanded CD133+/CD34+ cord blood fractions selected for transplantationare free of bacterial, yeast, mold and mycoplasm following initiation ofexpansion, on day 7, on day 14 and at the completion of expansion.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 5.6×10⁷ total CD34+ cells, at least 2.4×10⁷ total CD133+ cells, atleast 8×10⁷ total CD14+ cells, and at least 3.2×10⁷ total CD1(a and c)+cells. In further embodiments, the ex-vivo expanded CD133+/CD34+ cellsselected suitable for transplantation have at least 10×10⁸ total viablecells, at least 9% CD34+ cells, at least 5.6×10⁷ total CD34+ cells, atleast 4×10⁷ total CD133+ cells, at least 2×10⁸ total CD15+ cells, atleast 10×107 total CD11 b+ cells and at least 5×10⁷ total CD1(a and c)+cells.

In other embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 70% viability of the cells, at least 7% CD34+ cells, at least8×10⁵ CD133+/CD38− cells, at least 2×10⁸ total CD15+ cells and at least8×10⁷ total CD11b+ cells.

In further embodiments, the appearance of the ex-vivo expandedCD133+/CD34+ cord blood fraction is monitored. Observation of theexpanded CD133+/CD34+ cell fraction is directed to color and texture ofthe fraction. In some embodiments, ex-vivo expanded CD133+/CD34+ cellfractions appearing yellowish, cloudy-to opaque with no white clumps orforeign particles are selected.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation are washed and suspended in acryopreservation solution such as CryoStor®CS10 (BioLife Solutions.Inc.). The expanded CD133+/CD34+ fraction cell suspension is transferredinto the cryopreservation bag (CryoMACS freezing bag, Miltenyi Biotech),labeled and frozen using a control rate freezer. The storage is done inliquid nitrogen (LN).

In some embodiments, the first, ex-vivo cultured CD133+/CD34+ selectedcord blood fraction, and the second, unselected CD133/CD34 negative cordblood fraction are cryopreserved separately, in liquid nitrogen.

Transplantation of Expanded, Ex-vivo Cultured CD133+/CD34+ Selected CordBlood fractions and Unselected CD133/CD34 Negative Cord Blood Fractionsin a Subject

Expanded selected CD133+/CD34+ cord blood fractions, and unselectedCD133/CD34 negative fractions that have been selected suitable fortransplantation by can be used for transplantation into subjects in needthereof. Any of the methods for selection of cord blood units forex-vivo expansion described hereinabove, for expansion (ex-vivoculturing) of cord blood units described hereinabove, and/or fortransplantation following ex-vivo expansion, and/or for cryopreservationof the cord blood fractions, and each of their embodiments taken aloneor in various combinations may be used for affecting the methods forselecting cultured (ex-vivo expanded) CD133+/CD34+ selected andunselected. CD133/CD34 negative fractions as is described in thissection and the sections that follow. It will be appreciated that theex-vivo expanded CD133+/CD34+ and the uncultured CD133/CD34 negativecord blood cell fractions may be selected or excluded according to atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten or more, oraccording to all of the selection parameters disclosed herein.

In some embodiments, the subject in need of transplantation is sufferingfrom a hematological disease. In some embodiments, the subject issuffering from a hematological malignancy.

It will be appreciated that the methods, compositions, kits and articlesof manufacture of the present invention can be used to treat orameliorate symptoms of other indications, including but not limited tochronic myeloid leukemia, other myeloproliferative disorders, multiplemyeloma, aplastic anemia, pure red-cell aplasia, paroxysmal nocturnalhemoglobinuria, fanconi anemia, thalassemia major, sickle cell anemia,severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome,hemophagocytic lymphohistiocytosis, inborn errors of metabolism,epidermolysis bullosa, severe congenital neutropenia. Shwachman-Diamondsyndrome. Diamond-Blackfan anemia, leukocyte adhesion deficiency,autoimmune diseases and metabolic disease.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 2.4×10⁷ total CD133+ cells, at least 2×10⁸ total CD15+ cells, andat least 3.2×10⁷ total CD1(a and c)+ cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 83% viability of the cells, at least 2×10⁸ total CD14+ cells, atleast 1×10⁸ total CD11b+ cells and at least 6×10⁷ total CD1(a and c)+cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 83% viability of the cells, at least 2×10⁸ total CD14+ cells, atleast 1×10⁸ total CD11b+ cells and at least 6×10⁷ total CD1(a and c)+cells.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 70% viability, at least 8×10⁵total CD133+/CD38− cells and at least 8×10⁷ total CD11b+ cells.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 70% viability, at least 8×10⁵ total CD133+/CD38− cells and atleast 8×10⁷ total CD11b+ cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have a total CD34+ fraction of at least 7%,at least 8×10⁷ total CD14+ cells and at least 8×10⁷ total CD11b+ cells.

Subjects

As used herein, a “subject” or “patient” can be any mammal, e.g., ahuman, a primate, mouse, rat, dog, cat, cow, horse, pig, sheep, goat,camel. In a specific embodiment, the subject is a human.

As used herein, a “subject in need thereof” is a subject having the needfor transfusion, infusion or implantation of the cord blood fractions ofthe present invention to treat or ameliorate a disease, disorder orcondition. In one embodiment, the subject has (been diagnosed with) orsuffering from a hematological disease. In some embodiments, thehematological disease is a cell proliferative disorder. In otherembodiments, the hematological disease is a hematological malignancy.

As used herein, the term “risk of” or “probability of” refers to thelikelihood of an occurrence. In some embodiments, the risk orprobability of an occurrence (e.g. engraftment or non-engraftment of acord blood graft, non-relapse mortality, and the like) in an individualrefers to a risk calculated from comparative data between groupsreceiving treatment compared to groups not receiving the same treatment.In some embodiments, an increased or decreased risk or probabilityreflects the difference between treatment and control groups withrespect to the outcome under consideration. In some embodiments, anincrease or decrease in the risk or probability of a particularoccurrence or condition is only relative, and not expressed in numericalvalues.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. In specific embodiments, the methods provided hereinare used to treat or alleviate a symptom of cancer. The term “cancer”includes solid tumors, as well as, hematologic tumors and/ormalignancies. In preferred embodiments, the subject has (been diagnosedwith) or suffering from a hematologic malignancy. The hematologicmalignancy can be selected from the group consisting of acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), myelodysplastic syndrome (MDS),non-Hodgkin's lymphoma or Hodgkin's disease.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 75% viability of the cells, at least 7% CD34+ cells, at least8.1×10⁷ total CD34+ cells, at least 6×10⁷ total CD133+ cells, at least3×10⁸ total CD15+ cells, at least 8.7×10⁷ total CD11b+ cells and atleast 5×10⁷ total CD1(a and c)+ cells.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 7% CD34+ cells, at least 8×10⁵ CD133+/CD38− cells, at least 2×10⁸total CD15+ cells, and at least 8×10⁷ total CD11b+ cells.

In some embodiments, the methods and compositions and kits of thepresent invention can be used for treatment of subjects of all agegroups. In specific embodiments, the subject or patient is between 16-60years of age.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 2.4×10⁷ total CD133+ cells and at least 5.6×10⁷ total CD34+ cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 10×10⁸ total viable cells, atleast 83% viability of the cells and at least 7×10⁷ total CD1(a and c)+cells.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have a CD34+ cell fraction of at least 7%,at least 8×10⁵ total CD133+/CD38− cells and at least 8×10⁷ total CD14+cells.

In some embodiments, the subject in need thereof can have acutelymphoblastic leukemia (ALL) at one of the following stages: (a) highrisk first complete morphologic remission (CR1), defined as one or moreof the following: presence of t(4;11), t(9;22), t(1;19) or MLLrearrangements t(11q23); extreme leukocytosis (WBC >30,000/μl for B-ALLor >100.000/μl for T-ALL); Failure to achieve complete morphologicalremission after first induction therapy: Minimal Residual Disease (MRD)at screening by flow cytometry, or (b) second or subsequent remission.

The subject in need thereof can have acute myelogenous leukemia (AML) atone of the following stages: (a) First complete morphologic remission(CR1) that is not considered a favorable risk, favorable risk beingdefined as one or more of the following and absence of MRD at screening:t(8,21) without cKIT mutation; inv(16) or t(16;6) without cKIT mutation,normal karyotype with mutated NPM1 and no FLT-3 Internal TandemDuplication; normal karyotype with double mutated CEBPA; APL in first orsecond molecular remission at end of consolidation, or (b) second orsubsequent remission.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 70% viability of the cells, at least 8×10⁷ total CD14+ cells andat least 3.2×10⁷ total CD1(a and c)+ cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedto suitable for transplantation have a total CD34+ fraction of at least7%, at least 8×10⁷ total CD14+ cells and at least 8×10⁷ total CD11b+cells.

In further embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 83% viability of the cells, at least 2×10⁸ total CD14+ cells, atleast 1×10⁸ total CD11b+ cells and at least 6×10⁷ total CD1(a and c)+cells.

The subject in need thereof can have chronic myelogenous leukemia (CML)at one of the following stages: (a) Chronic phase with one or more ofthe following characteristics: Failure to achieve a rimaryhematologicalor cytogenic response to either nilotinib or dasatinib (followingEuropean Leukemia Net timelines). Intolerance to/failure of two tyrosinekinase inhibitors (TKI), and any T315I mutation: (b) Accelerated phasewith one or more of the following characteristics: Newly diagnosedpatient not achieving optimal response to TKIs as outlined in EuropeanLeukemiaNet timelines: TKI patients progressing from chronic phase, or(c) a Prior blast crisis (myeloid and lymphoid) currently in completemorphological remission.

The subject in need thereof can have Myelodysplastic Syndrome (MDS) withInternational Prognostic Scoring System (IPSS) risk category of INT-1 orgreater. On screening morphologic analysis patients have no circulatingmyeloblasts and ≤10% myeloblasts in the bone marrow. MDS patientscategorized as INT-1 on primary presentation have life threateningneutropenia (ANC <0.5×109/L) or thrombocytopenia (platelets <30×109/L).

The subject in need thereof can have Non-Hodgkin's lymphoma or Hodgkin'sdisease with at least one of the following features: (a) progressed ornon-responsive to upfront chemotherapy and has achieved a partialremission (partial remission defined as ≥50% reduction of disease fromthe prior course of chemotherapy) to subsequent therapy or (b) second orsubsequent complete or partial remission (partial remission defined as≥50% reduction of disease from the prior course of chemotherapy) and notappropriate candidates for autologous stem cell transplantation.

In some embodiments, the ex-vivo expanded CD133+/CD34+ cells selectedsuitable for transplantation have at least 70% viability of the cells,at least 7% CD34+ cells, at least 2.4×10⁷ total CD133+ cells, at least8×10⁷ total CD14+ cells and at least 3.2×10⁷ total CD1(a and c)+ cells.In some embodiments, the ex-vivo expanded to CD133+/CD34+ cells selectedsuitable for transplantation have at least 8×10⁸ total viable cells, atleast 2×10⁸ total CD15+ cells and at least 8×10⁷ total CD11b+ cells.

In some embodiments, a subject in need thereof can be defined accordingto the following criteria: a performance score of at least 70% byKarnofsky, and sufficient physiologic reserves including: a. Cardiac:Left ventricular ejection fraction (LVEF) of ≥40% by echocardiogram,radionuclide scan or cardiac MRI; b. Pulmonary function testsdemonstrating FVC and FEV1 of >50% of predicted for age and cDLCO>50% ofpredicted; c. Renal: Creatinine clearance test (by Cockcroft-Gaultequation) ≥60 mL/min, and d. Hepatic: Serum Bilirubin <2.0 mg/dl;Hepatic transaminases (ALT and AST)<3×upper limit of normal range

In some embodiments, subjects can be excluded from consideration fortreatment for any of the following:

-   -   1. MDS or CML with “marked” or “3+” fibrosis;    -   2. CMMoL or MDS/CMMoL overlap;    -   3. Fewer than 21 days elapsed since initiation of the patient's        last chemotherapy cycle and the initiation of the stem cell        transplant preparative regimen (intrathecal agents, hydroxyurea,        tyrosine kinase inhibitors, hypomethylating agents, rituximab        and lenalidomide not considered chemotherapy);    -   4. Persistent clinically significant toxicities that make the        patient unsuitable for transplant;    -   5. Evidence of anti-HLA antibodies to a candidate cord blood        unit profile (MFI>3000 to HLA A, B, C, or DRB1);    -   6. Evidence of HIV infection or HIV positive serology;    -   7. Evidence of active Hepatitis B, Hepatitis C or EBV as        determined by serology or PCR;    -   8. Pregnancy, as indicated by a positive serum human chorionic        gonadotrophin (HCG) test, or lactation;    -   9. Active malignancy other than that for which the UCB        transplant is being performed within 12 months of enrollment in        treatment program. Fully resected cutaneous squamous cell or        basal cell carcinoma or cervical carcinoma in situ within 12        months of enrollment is permitted;    -   10. Evidence of uncontrolled bacterial, fungal or viral        infections or severe concomitant diseases, which indicate that        the patient could not tolerate transplantation;    -   11. Patients with signs and symptoms of leukemic blasts in the        central nervous system (CNS);    -   12. Patients with an 8/8 allele level HLA-matched and readily        available related or unrelated donor (whose stem cells can be        collected in a timely manner without jeopardizing recipient        outcome);    -   13. Prior allogeneic hematopoietic stem cell transplant;    -   14. Allergy to bovine products, gentamicin, or to any other        product that may interfere with the treatment;    -   15. Psychologically incapable of undergoing bone marrow        transplant (BMT) with associated strict isolation or documented        history of medical non-compliance and/or psychiatric illness        and/or social situations that would limit compliance with        treatment requirements, and    -   16. Enrolled in an interventional clinical trial or received an        investigational treatment within 30 days prior to the approved        date of treatment.

In some embodiments, the subject in need thereof has been subjected tomyeloablative therapy or conditioning regime. In specific embodiments,the subject has been subjected to myeloablative therapy or conditioningregime prior to transplantation or administration of the compositions ofthe present invention. The subject can be subjected to the myeloablativetherapy or conditioning regime between 11 days (−11) to 2 days (−2)prior to transplantation or administration of the compositions of thepresent invention, or, in another regimen, between 8 days (−8) to 1 day(−1) prior to transplantation or administration of the compositions ofthe present invention, or between 7 days (−7) to 3 days (−3) prior totransplantation or administration of the compositions of the presentinvention. The myeloablative therapy or conditioning regime can includetotal body irradiation (TBI) and fludarabine, and may also includeBusulfan, cyclophosphamide or Thiotepa. In one regimen, total radiationdose will be 1350 cGy in 8 or 9 fractions over 5 days. Two fractions onthe same day will be given at a minimum of 6 hours apart from beam on tobeam on, for example, on days −9 to −5 of the 11-day regimen. In the 11day regimen, fludarabine (40 mg/m²/day) will be administered IV, forexample, on days −5 to −2. Fludarabine will be dosed as per adjustedideal body weight. In the 11 day regimen, Thiotepa (5 mg/kg) isadministered IV on days −11 and −10.

In an 8 day regimen, total radiation dose will be 1350 cGy in 8fractions over 4 days, two fractions on the same day will be given at aminimum of 6 hours apart from beam on to beam on, on days −4 to −1.Fludarabine 25 mg/m²/day is administered over 60 minutes IV infusion ondays −8 through −6. Fludarabine will be dosed as per adjusted ideal bodyweight. In some embodiments, the regime can also includecyclophosphamide. Cyclophosphamide can be administered at 60 mg/Kg/dayas per adjusted ideal body weight, for example, on days −8 and −7 of the8 day regimen.

Some conditioning regimen do not include TBI. In an exemplary 7 dayregimen, Thiotepa (5 ng/kg) is administered intravenously on days −7 and−6, and Busulfan (3.2 mg/kg or adjusted for cumulative Busulfan exposureof 75 mg*h/L after 3 days) is administered intravenously on days −5 to−4. In such a regimen, fludarabine 50 mg/m²/day is administered over 60minutes in IV infusions on days −5 through −3.

In some embodiments, the subject in need thereof has been subjected tograft-versus-host disease (GvHD) prophylaxis regime. In one embodiment,the subject has been subjected to GvHD prophylaxis regime prior totransplantation or administration of the compositions of the presentinvention. The subject can be subjected to the GvHD prophylaxis regimebetween 9 days (−9) to 1 day (−1) prior to transplantation oradministration of the compositions of the present invention. In otherembodiments, the subject has the GvHD prophylaxis regime at least 3 daysprior (−3) to transplantation or administration of the compositions ofthe present invention, to at least day +150. The subject can besubjected to either myeloablative therapy or conditioning regime or GvHDprophylaxis regime individually, or in combination. The GvHD prophylaxisregime can include mycophenolate mofetil (MMF) and tacrolimus. In someembodiments, tacrolimus is administered via IV from day −3 until oralmedications are tolerated, then PO through day +150 to target bloodlevel of 5-15 ng/ml and MMF is administered 1 g TID via IV or PO (15mg/kg IV TID if patient weighs <50 kg) beginning day −3 to at least day+60.

In some embodiments, subjects in need thereof will have an appropriatelong-term central venous access placed prior to the conditioningregimen. In some embodiments, the subjects will have a triple lumentunneled catheter.

In some embodiments, subjects can receive any or all of the following:infusion support (e.g. diphenylhydramine or dexchlorpheniramine,hydrocortisone and acetaminophen), supportive cytokines (e.g. G-CSF),blood products as needed, anti-viral, anti-bacterial, PCP and/or fungalprophylaxis, CMV, EBV and HHV6 surveillance and IV immunoglobulin asneeded.

In some embodiments, subjects receive any or all of an additionaltreatment for the hematological disease. Said treatment can be atreatment selected from the group consisting of an immunosuppressivetreatment, chemotherapy and radio-therapy.

Thus, in some embodiments there is provided a method of treating ahematological disease in a subject in need thereof, the methodcomprising:

-   -   (a) separating a single umbilical cord blood unit suitable for        transplantation into said subject into (i) a first, selected        blood cell fraction comprising CD133+/CD34+ selected cells        and (ii) a second, unselected blood cell fraction comprising        CD133/CD34 negative cells;    -   (b) ex vivo culturing said first blood cell fraction comprising        CD133+/CD34+ selected cells under conditions allowing for cell        proliferation, said conditions comprising providing nutrients,        serum and a combination of cytokines including each of stem cell        factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in        an amount between 1.0 mM to 10 mM;    -   (c) cryopreserving said ex-vivo cultured first, CD133+/CD34+        selected blood cell fraction and said uncultured second,        unselected blood cell fraction from step (b),    -   (d) thawing said ex-vivo cultured first selected and said        uncultured second, unselected blood cell fractions, and    -   (e) transplanting the thawed ex-vivo cultured first selected and        uncultured second unselected blood cell fractions from step (d)        into a subject in need thereof, thereby treating said        hematological disease in said subject.

In some embodiments, the method of treating a hematological disease in asubject in need thereof consists of:

-   -   (a) separating a single umbilical cord blood unit suitable for        transplantation into said subject into (i) a first, selected        blood cell fraction comprising CD133+/CD34+ selected cells        and (ii) a second, unselected blood cell fraction comprising        CD133/CD34 negative cells;    -   (b) ex vivo culturing said first blood cell fraction comprising        CD133+/CD34+ selected cells under conditions allowing for cell        proliferation, said conditions comprising providing nutrients,        serum and a combination of cytokines including each of stem cell        factor, thrombopoietin. FLt3 ligand and IL-6 and nicotinamide in        an amount between 1.0 mM to 10 mM;    -   (c) cryopreserving said ex-vivo cultured first. CD133+/CD34+        selected blood cell fraction and said uncultured second,        unselected blood cell fraction from step (b),    -   (d) thawing said ex-vivo cultured first selected and said        uncultured second, unselected blood cell fractions, and    -   (e) transplanting the thawed ex-vivo cultured first selected and        uncultured second unselected blood cell fractions from step (d)        into a subject in need thereof, thereby treating said        hematological disease in said subject.

In some embodiments, the single umbilical cord blood unit suitable fortransplantation is characterized by the following pre-cryopreservationparameters:

-   -   (i) at least 8×10⁶ total CD34+ cells;    -   (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A and        HLA-B, low resolution) and HLA class II (HLA-DRB1, high        resolution) loci with said subject;    -   (iii) about 1.8×10⁹ to about 3.0×10⁹ pre-cryopreserved total        nucleated cells, and    -   (iv) about 1.5×10⁷ to about 3.0×10⁷ pre-cryopreserved total        nucleated cells per kilogram subject weight.

In a particular embodiment, the first cryopreserved ex-vivo culturedselected and said uncultured second, unselected blood cell fractions arethawed on the same day of transplantation. In some embodiments, Thefirst cryopreserved ex-vivo cultured (expanded) CD133+/CD34+ fraction iskept frozen until the day of transplantation. On the day oftransplantation, at the clinical site, first cryopreserved ex-vivocultured (expanded) CD133+/CD34+ fraction is thawed and reconstitutedwith the infusion solution (8% w/v Human Serum Albumin (HSA) and 6.8%w/v Dextran-40) in a closed system.

In some embodiments, the unselected, CD133/CD34 negative cord bloodfraction of the same cord blood unit is the fraction of cells which iseluted during the separation (e.g. CliniMACS positive separation) ofCD133⁺ cells. This fraction contains the entire repertory of immunecells, such as B, T and NK cells. The cells are washed and suspended incryopreservation solution, CryoStor®CS10 (BioLife Solutions, Inc.) afterelution from the selection column (e.g. CliniMACS column), labeled andthen frozen using a control rate freezer. The storage is done in liquidnitrogen. The unselected CD133/CD34 negative fraction is kept frozenuntil the day of transplantation into the subject. On the day oftransplantation, the unselected, CD133/CD34 negative fraction is thawedand reconstituted with the infusion solution (8% w/v HSA and 6.8% w/vDextran-40) in a closed system. In some embodiments, the infusionsolution is screened for suitability for use with the methods andcompositions of the present invention. Exemplary criteria for selectionof suitable infusion solution include safety tests indicating nobacterial, yeast or mold growth, endotoxin content of less than 0.5Eu/ml and a clear, foreign particle-free appearance.

In some embodiments, the infusion solution is stored in bags until use(e.g. transplantation) at 2-8° C.

In some specific embodiments, transplantation of cord blood fractions ispreceded by a safety assessment of the subject in need thereof on theday of transplantation, typically including a physical examination. CBC,blood chemistry (e.g. at least serum creatinine, total bilirubin,alkaline phosphatase, AST, ALT and magnesium), Vital Signs: weight,temperature, blood pressure, pulse, and respiratory rate, andadministration of concomitant medication, including RBC and platelettransfusions.

Infusion of the selected, expanded CD133+CD34+ and the unselected.CD133CD34 negative cord blood fractions into the subject in need thereofis typically done by transfusion via the patient's central venouscatheter, subject to the limitations of individual site practice. Insome embodiments, the selected, expanded CD133+/CD34+ cord bloodfraction is infused first, followed immediately by the infusion of theunselected, CD133/CD34 negative (unmanipulated) cord blood fraction. Inother embodiments, the unselected, CD133/CD34 negative (unmanipulated)cord blood fraction is infused first, followed immediately by theinfusion of the selected, expanded CD133+/CD34+ cord blood fraction.

Thus, in some embodiments, of the present invention, transplantation isaffected by infusion in an infusion solution into said patient, whereinsaid ex-vivo cultured first selected blood cell fraction is infusedprior to said uncultured second, unselected blood cell fraction, whilein other embodiments uncultured the second, unselected blood cellfraction is infused prior to said ex-vivo cultured first selected bloodcell fraction.

As detailed hereinabove, both the selected, expanded CD133+/CD34+ cordblood fraction and the unselected. CD133/CD34 negative (unmanipulated)cord blood fraction selected suitable for transplantation are screened,inter alia, for total viable cell content. In some embodiments, thefirst ex-vivo cultured CD133+CD34+ blood cell fraction for infusion intothe subject comprises at least 8×10⁸ total viable cells. In someembodiments the second uncultured CD133/CD34 negative blood cellfraction for infusion into the subject comprises at least 4×10⁸ totalviable cells.

The method of treatment of hematological disease of the presentinvention can be used to treat hematological malignancies, including,but not limited to Chronic myelogenous leukemia (CML), acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) andmyelodysplastic syndrome (MDS). Also contemplated is treatment ofHodgkin's Disease and non-Hodgkin's Lymphoma. As used herein, the term“treating a hematological disease” or “treating a hematologicalmalignancy” refers to reducing the symptoms or signs of thehematological disease. In some embodiments, treating hematologicaldiseases or a hematological malignancy is assessed according to, but notexclusively, reduction in symptoms over time, improvement in clinicalparameters such as neutrophil and platelet count, reducedhospitalization and reduced risk of relapse or mortality.

Thus, according to some embodiments of the method of the presentinvention, separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin. FLt3ligand and TL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first. CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases time from transplantation to neutrophilengraftment in said subject, when compared to transplantation of asingle or double unit of unmanipulated cord blood.

As used herein, the term “unmanipulated cord blood” refers to cord bloodthat has not undergone selection, and or expansion as describedherewith. In some embodiments, unmanipulated cord blood is a fresh unitor a cryopreserved cord blood unit from the cord blood bank.

In some embodiments, transplantation of said first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood decreases the time to neutrophil engraftment by 5-14 days whencompared to transplantation of a single or double unit of unmanipulatedcord blood.

As used herein, the term “neutrophil engraftment” refers to achieving anabsolute neutrophil count (ANC) ≥0.5×10⁹/L on 3 consecutive measurementson different days with subsequent donor chimerism (≥10% host cells byperipheral blood chimerism), on or before 42 days post transplant (andprior to infusion of any additional stem cells product). In someembodiments, decreasing the time to neutrophil engraftment refers toachieving an absolute neutrophil count (ANC) ≥0.5×10⁹/L on 3 consecutivemeasurements on different days with subsequent donor chimerism (≤10%host cells by peripheral blood chimerism), on or before 16 days posttransplant.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin, FLt3ligand and TL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first. CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases the time from transplantation to plateletengraftment in said subject, when compared to transplantation of asingle or double unit of unmanipulated cord blood.

In other embodiments, transplantation of said first ex vivo culturedCD133+/CD34+ hematopoietic stein/progenitor blood cell fraction and saidsecond uncultured CD133−/CD34− blood cell fraction increases theprobability of platelet engraftment in said subject at 42 days posttransplantation when compared to transplantation of a single or doubleunit of unmanipulated cord blood.

In specific embodiments, the term “platelet engraftment” relates to thefirst day of a minimum of 3 consecutive measurements on different dayssuch that the patient has achieved a platelet count >20×109/L with noplatelet transfusions in the preceding 7 days (count day of engraftmentas one of the preceding 7 days). The first day of the three measurementswill be designated the day of platelet engraftment and occurs prior toany infusion of a second stein cell product.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin, FLt3ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM:cryopreserving said ex-vivo cultured first, CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases the probability of non-engraftment by day 42after transplantation in said subject, when compared to transplantationof a single or double unit of unmanipulated cord blood. In specificembodiments, the term “primary graft failure”, or “non-engraftment”relates to failure to achieve neutrophil engraftment by day 42, asdescribed hereinabove. Requirement for infusion of a second stem cellproduct to the subject on or prior to day 42 following transplantationis also considered primary graft failure. However, need for infusion ofan additional stem cell product after documented neutrophil engraftmentcan be considered secondary graft failure, even if it occurs on or priorto day 42 post transplantation. The date of primary graft failure willbe designated as day 43 post transplant.

In other embodiments, transplantation of said first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction into a subject in need thereof according to themethods of the present invention decreases the probability of secondarygraft failure or secondary non engraftment when compared totransplantation of a single or double unit of unmanipulated cord blood.In specific embodiments, the term “secondary graft failure”, or“non-engraftment” relates to documented neutrophil engraftment, followedby severe neutropenia (<0.5×109/L for three or more consecutivelaboratory values on separate days) with marrow cellularity <5%, withoutsubsequent improvement occurring either spontaneously or after growthfactor treatment. Infusion of an additional stem cell product afterdocumented neutrophil engraftment is considered secondary graft failure.The earlier of the first day of severe neutropenia, as defined above, orthe date of additional stem cell infusion will be designated the date ofsecondary graft failure.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin. FLt3ligand and TL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first. CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases the probability of non-relapse mortality aftertransplantation in said subject, when compared to transplantation of asingle or double unit of unmanipulated cord blood. As used herein, theterm “non-relapse mortality” refers to any death not preceded by relapseof the subject's hematological disease or diseases.

In some embodiments, transplantation of said first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction decreases the probability of non-relapse mortalityat 180-210 days after transplantation, when compared to transplantationof a single or double unit of unmanipulated cord blood.

In some embodiments, transplantation of said first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction decreases the risk of non-relapse mortality at 1year when compared to transplantation of a single or double unit ofunmanipulated cord blood. In some embodiments, transplantation of saidfirst ex vivo cultured CD133+/CD34+ blood cell fraction and said seconduncultured CD133−/CD34− blood cell fraction according to the methods ofthe present invention decreases the risk of non-relapse mortality at 15months post transplantation year when compared to transplantation of asingle or double unit of unmanipulated cord blood.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin. FLt3ligand and TL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first. CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases duration of hospitalization in said subject inthe first 100 days post-transplantation, when compared totransplantation of a single or double unit of unmanipulated cord blood.As used herein, the term “duration of hospitalization” refers to thetotal number of days from transplant to the first discharge from thehospital.

In some embodiments, transplantation of the first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction decreases duration of post-transplantationhospitalization by 5-30 days, when compared to transplantation of asingle or double unit of unmanipulated cord blood. In some embodiments,transplantation of the first ex vivo cultured CD133+/CD34+ blood cellfraction and said second uncultured CD133−/CD34− blood cell fractionincreases the number of days alive and out of hospital followingtransplantation, when compared to transplantation of a single or doubleunit of unmanipulated cord blood. Days alive and out of the hospital, asused herein, are defined as a full days (calendar days) in which thepatient was alive and not hospitalized.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin. FLt3ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first, CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases risk of grade 2/3 bacterial or invasive fungalinfections post-transplantation in said subject, when compared totransplantation of a single or double unit of unmanipulated cord blood.Grading of bacterial and/or invasive fungal infections is well known inthe art. Signs of Grade 3 bacterial infections include, but are notlimited to Bacteremia with deep organ involvement (e.g. with new orworsening pulmonary infiltrates; endocarditis), Severe sepsis withbacteremia.

Fasciitis requiring debridement. Pneumonia requiring intubation. Brainabscess or meningitis without bacteremia and Difficile toxin positivestool with toxic dilatation or renal insufficiency with/withoutdiarrhea. Signs of Grade 3 fungal infections include, but are notlimited to Fungemia including Candidemia, Proven or probable invasivefungal infections (e.g., Aspergillus, Mucor, Fusarium, Scedosporium),Disseminated infections (defined as multifocal pneumonia, presence ofurinary or blood antigen, and/or CNS involvement) with Histoplasmosis,Blastomycosis, Coccidiomycosis, or Cryptococcus and Pneumocystisjirovecii pneumonia (regardless of PaO2 level).

In some embodiments, transplantation of the first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction according to the methods of the present inventiondecreases risk of grade 2/3 bacterial or invasive fungal infectionspost-transplantation decreases risk of grade 2/3 bacterial or invasivefungal infections by 100 days post-transplantation in said subject, whencompared to transplantation of a single or double unit of unmanipulatedcord blood.

According to some embodiments of the method of the present invention,separating a single umbilical cord blood unit suitable fortransplantation into said subject into (i) a first, selected blood cellfraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; exvivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin. FLt3ligand and TL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;cryopreserving said ex-vivo cultured first. CD133+/CD34+ selected bloodcell fraction and said uncultured second, unselected blood cellfraction; thawing said ex-vivo cultured CD133+/CD34+ first selected andsaid uncultured CD133/CD34 negative second, unselected blood cellfractions, and transplanting the thawed ex-vivo cultured first selectedand uncultured second unselected blood cell fractions into a subject inneed thereof decreases the risk of acute graft-versus-host disease gradeIII-IV in said subject at 100 days post transplantation, when comparedto transplantation of a single or double unit of unmanipulated cordblood. In some embodiments, transplantation of the first ex vivocultured CD133+/CD34+ blood cell fraction and said second unculturedCD133−/CD34− blood cell fraction according to the methods of the presentinvention decreases the risk of acute graft-versus-host disease gradeIU-IV in said subject at 180 days post transplantation in said subject,when compared to transplantation of a single or double unit ofunmanipulated cord blood.

In some embodiments, transplantation of the first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction according to the methods of the present inventiondecreases the risk of chronic graft-versus-host disease(mild/moderate/severe) in said subject at 180 days or 1 year posttransplantation in said subject, when compared to transplantation of asingle or double unit of unmanipulated cord blood.

As used herein the term “acute GvHD” refers to acute Graft versus HostDisease, occurring earlier following transplantation, and chronic orcGvHD refers to chronic Graft versus Host Disease, present later, afterthe cut off point of Acute GvHD post-transplantation. In someembodiments, acute GvHD is classified according to the ConsensusConference on Acute GvHD grading (see Przepiorka et al, Bone MarrowTransplant 1995, 15:825-828), and cGvHD is classified according to theNIH consensus criteria for grading cGvHD (see Jagasia et al, Biol. BloodMarrow Transplant, 2015 21:389-401).

In some embodiments, of the method of the present invention, separatinga single umbilical cord blood unit suitable for transplantation intosaid subject into (i) a first, selected blood cell fraction comprisingCD133+/CD34+ selected cells and (ii) a second, unselected blood cellfraction comprising CD133/CD34 negative cells; ex vivo culturing saidfirst blood cell fraction comprising CD133+/CD34+ selected cells underconditions allowing for cell proliferation, said conditions comprisingproviding nutrients, serum and a combination of cytokines including eachof stem cell factor, thrombopoietin. FLt3 ligand and IL-6 andnicotinamide in an amount between 1.0 mM to 10 mM; cryopreserving saidex-vivo cultured first. CD133+/CD34+ selected blood cell fraction andsaid uncultured second, unselected blood cell fraction; thawing saidex-vivo cultured CD133+/CD34+ first selected and said unculturedCD133/CD34 negative second, unselected blood cell fractions, andtransplanting the thawed ex-vivo cultured first selected and unculturedsecond unselected blood cell fractions into a subject in need thereofincreases the probability of disease-free survival by 15 months posttransplantation, increases the probability of overall survival by 210days, and/or 15 months post transplantation, improves immunereconstitution (numbers and proportions of different lymphocytesubpopulations) in said subject, for example, by 28, 70, 100, 180 and365 days post transplantation, when compared to transplantation of asingle or double unit of unmanipulated cord blood.

In some embodiments of the method of the present invention, separating asingle umbilical cord blood unit suitable for transplantation into saidsubject into (i) a first, selected blood cell fraction comprisingCD133+/CD34+ selected cells and (ii) a second, unselected blood cellfraction comprising CD133/CD34 negative cells; ex vivo culturing saidfirst blood cell fraction comprising CD133+/CD34+ selected cells underconditions allowing for cell proliferation, said conditions comprisingproviding nutrients, serum and a combination of cytokines including eachof stem cell factor, thrombopoietin, FLt3 ligand and TL-6 andnicotinamide in an amount between 1.0 mM to 10 mM; cryopreserving saidex-vivo cultured first, CD133+/CD34+ selected blood cell fraction andsaid uncultured second, unselected blood cell fraction; thawing saidex-vivo cultured CD133+/CD34+ first selected and said unculturedCD133/CD34 negative second, unselected blood cell fractions, andtransplanting the thawed ex-vivo cultured first selected and unculturedsecond unselected blood cell fractions into a subject in need thereofdecreases the probability of disease relapse and relapse mortality insaid subject post transplantation, when compared to transplantation of asingle or double unit of unmanipulated cord blood.

In some embodiments, disease relapse is defined according to thefollowing criteria:

Relapse of malignancy—Relapse is defined by either morphological orcytogenetic evidence of AML, ALL, CML, or MDS consistent withpre-transplant features.

Minimal residual disease—minimal residual disease (MRD) is defined bythe sole evidence of malignant cells by flow cytometry, or fluorescentin situ hybridization (FISH), or Southern blot or Western blot, orpolymerase chain reaction (PCR), or other techniques, in the absence ofmorphological or cytogenetic evidence of disease in blood or marrow.Evidence of minimal residual disease alone is not sufficient to meet thedefinition of relapse, however, minimal residual disease that progressescan be considered as relapse.

Acute Leukemia—Relapse can be defined as any of the following:

-   -   >5% blasts in the marrow, not attributed to other causes (e.g.,        bone marrow regeneration) The appearance of new dysplastic        changes within the bone marrow, not attributed to other causes.

Reappearance of leukemic blasts in the peripheral blood.

Reappearance of previous cytogenetic or molecular marker of diseasepresent prior to transplantation.

The development of extramedullary leukemia or leukemic cells in thecerebral spinal fluid.

Institution of any therapy in response to detection of MRD, includingwithdrawal of immunosuppressive therapy or the addition of tyrosinekinase inhibitors or hypomethylating agents, will be considered evidenceof relapse regardless of whether the criteria described above are met.

Chronic Myelogenous Leukemia (CML)

Hematologic relapse can be diagnosed when:

-   -   1. Immature hematopoietic cells are persistently documented in        the peripheral blood; or.    -   2. There is myeloid hyperplasia in the bone marrow in the        presence of cytogenetic relapse.

Cytogenetic relapse can be diagnosed when:

-   -   1. 50% of the metaphases exhibit the characteristic (9;22)        translocation with at least ten metaphases analyzed; or,    -   2. At least one metaphase exhibits the (9;22) translocation on        each of two separate consecutive examinations at least one month        apart, regardless of number of metaphases analyzed. The date of        the earliest test will be considered the date of relapse.

MDS

Relapse can be defined as any of the following:

-   -   Satisfying above criteria for evolution into acute leukemia: or,    -   Reappearance of pre-transplant morphologic abnormalities,        detected in two consecutive bone marrow specimens taken at least        one month apart (the date of the earliest test will be        considered the date of relapse); or,    -   Reappearance of pre-transplant cytogenetic abnormality in at        least one metaphase on each of two separate consecutive        examinations at least one month apart, regardless of the number        of metaphases analyzed.

Institution of any therapy in response to detection of MRD, includingwithdrawal of immunosuppressive therapy or treatment withhypomethylating agents, is considered evidence of relapse regardless ofwhether the criteria described above are met.

In some embodiments, the present invention provides an article ofmanufacture, a composition or kit comprising a packaging material andumbilical cord blood cell fractions comprising:

-   -   (i) a first blood cell fraction suitable for transplantation        into a subject in need thereof comprising CD133+/CD34+ selected        cord blood cells ex-vivo cultured under conditions allowing for        cell proliferation, said conditions comprising providing        nutrients, serum and a combination of cytokines including each        of stem cell factor, thrombopoietin, FLt3 ligand and IL-6 and        nicotinamide in an amount between 1.0 mM to 10 mM; and (ii) a        second, uncultured, unselected blood cell fraction suitable for        transplantation into a subject in need thereof comprising        CD133/CD34 negative cells, wherein said packaging material        comprises a label or package insert indicating that said first        and second umbilical cord blood cell populations are for        treating a hematological disease in a subject in need thereof.

In some embodiments, the article of manufacture, composition or kit ofthe present invention further comprises instructions for administeringthe (i) first blood cell fraction suitable for transplantation into asubject in need thereof comprising to CD133+/CD34+ selected cord bloodcells and (ii) the second, uncultured, unselected blood cell fractionsuitable for transplantation into a subject in need thereof comprisingCD133/CD34 negative cells to a subject in need thereof.

In some embodiments of the article of manufacture, composition or kit ofthe present invention, the (i) first blood cell fraction suitable fortransplantation into a subject in need thereof comprising CD133+/CD34+selected cord blood cells comprises at least 8×10⁸ total viable cells.In some embodiments, the first CD133+/CD34+ selected cord blood fractioncomprises at least 10×10⁸ total viable cells, at least 15×10⁸ totalviable cells, at least 20×10⁸ total viable cells or at least 25×10⁸total viable cells. In some embodiments of the article of manufacture,composition or kit of the present invention, the (ii) second,uncultured, unselected blood cell fraction suitable for transplantationinto a subject in need thereof comprising CD133/CD34 negative cellscomprises 4×10⁸ total viable cells. In some embodiments, second,uncultured, unselected blood cell fraction suitable for transplantationinto a subject in need thereof comprising CD133/CD34 negative cellscomprises at least 6×10⁸ total viable cells, at least 8×10⁸ total viablecells, at least 10×10⁸ total viable cells or at least 15×10⁸ totalviable cells.

In some embodiments of the article of manufacture, composition or kit ofthe present invention, the (i) first blood cell fraction suitable fortransplantation into a subject in need thereof comprising CD133+/CD34+selected cord blood cells and the (ii) second, uncultured, unselectedblood cell fraction suitable for transplantation into a subject in needthereof comprising CD133/CD34 negative cells are provided ascryopreserved cord blood cell fractions.

Selected cell populations of the present invention can be provided perse, along with the culture medium containing same, isolated from theculture medium, and combined with a pharmaceutically acceptable carrieras well as with additional agents which may promote cell engraftmentand/or organ function (e.g., immunosuppressing agents, antibiotics,growth factor). Hence, cell populations of the invention can beadministered in a pharmaceutically acceptable carrier or diluent, suchas sterile saline and aqueous buffer solutions. The use of such carriersand diluents is well known in the art.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit or article ofmanufacture, which may contain one or more unit dosage forms containingthe active ingredient (e.g., cells). The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser device may also be accompanied by a notice in a formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions for human or veterinaryadministration. Such notice, for example, may include labeling approvedby the U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a pharmaceutically acceptable carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition, as further detailed above.

The cells prepared according to the methods of the present invention canbe administered to the subject per se, or in a pharmaceuticalcomposition where it is mixed with suitable carriers or excipients.

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier.” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, PA, which is herein fully incorporated byreference.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., expanded CD133+/CD34+ cells) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,leukemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E, et al. (1975). “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations.The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination to in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

1. A method of selecting a cryopreserved umbilical cord blood unit forex-vivo expansion and transplantation into a subject, comprising: (a)determining in said umbilical cord blood unit prior to ex-vivo expansionthe following pre-cryopreservation parameters: (i) about 8×106 to about15×106 total CD34+ cells; (ii) HLA-matched at at least 4 out of 6 HLAclass I (HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1,high resolution) loci with said subject; (iii) about 1.8×109 to about3.0×109 pre-cryopreserved total nucleated cells; (iv) about 1.5×107 toabout 3.0×107 pre-cryopreserved total nucleated cells per kilogramsubject weight, (b) selecting or excluding said umbilical cord bloodunit according to said parameters, thereby selecting an umbilical cordblood unit suitable for ex-vivo expansion and transplantation into thesubject.
 2. The method of claim 1, wherein (i) is about 8×106 to about10×106 total CD34+ cells.
 3. (canceled)
 4. A method of treating ahematological disease in a subject in need thereof, the methodcomprising: (a) separating a single umbilical cord blood unit suitablefor transplantation into said subject into (i) a first, selected bloodcell fraction comprising CD133+/CD34+ selected cells and (ii) a second,unselected blood cell fraction comprising CD133/CD34 negative cells; (b)ex vivo culturing said first blood cell fraction comprising CD133+/CD34+selected cells under conditions allowing for cell proliferation, saidconditions comprising providing nutrients, serum and a combination ofcytokines including each of stem cell factor, thrombopoietin, FLt3ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;(c) cryopreserving said ex-vivo cultured first, CD133+/CD34+ selectedblood cell fraction and said uncultured second, unselected blood cellfraction from step (b), (d) thawing said ex-vivo cultured first selectedand said uncultured second, unselected blood cell fractions, and (e)transplanting the thawed ex-vivo cultured first selected and unculturedsecond unselected blood cell fractions from step (d) into a subject inneed thereof, thereby treating said hematological disease in saidsubject, wherein said single umbilical cord blood unit suitable fortransplantation is characterized by the following pre-cryopreservationparameters: (i) at least 8×106 total CD34+ cells; (ii) HLA-matched at atleast 4 out of 6 HLA class I (HLA-A and HLA-B, low resolution) and HLAclass II (HLA-DRB1, high resolution) loci with said subject; (iii) about1.8×109 to about 3.0×109 pre-cryopreserved total nucleated cells, and(iv) about 1.5×107 to about 3.0×107 pre-cryopreserved total nucleatedcells per kilogram subject weight. 5-6. (canceled)
 7. The method ofclaim 4, wherein said cryopreserved ex-vivo cultured first selected andsaid uncultured second, unselected blood cell fractions are thawed onthe same day of transplantation.
 8. The method of claim 4, wherein saidcryopreserved ex-vivo cultured first selected and said unculturedsecond, unselected blood cell fractions are thawed and reconstituted ininfusion solution and wherein said transplantation is affected byinfusion in an infusion solution into said patient. 9-10. (canceled) 11.The method of claim 8, wherein said ex-vivo cultured first selectedblood cell fraction is infused prior to said uncultured second,unselected blood cell fraction.
 12. The method of claim 8, wherein saiduncultured second, unselected blood cell fraction is infused prior tosaid ex-vivo cultured first selected blood cell fraction.
 13. The methodof claim 4, wherein said first ex-vivo cultured blood cell fractioncomprises at least 8×108 total viable cells and said second unculturedblood cell fraction comprises at least 4×108 total viable cells. 14.(canceled)
 15. The method of claim 4, wherein said hematological diseaseis a hematological malignancy selected from the group consisting ofchronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). 16.(canceled)
 17. The method claim 15, wherein the subject has received atleast one treatment selected from the group consisting of: (a)myeloablative therapy or other conditioning regime prior totransplantation; (b) Graft-versus-host disease (GvHD) prophylaxis regimeprior to transplantation; and (c) Graft-versus-host disease (GvHD)prophylaxis regime following transplantation. 18-19. (canceled)
 20. Themethod of claim 4, wherein transplantation of said first ex vivocultured CD133+/CD34+ blood cell fraction and said second unculturedCD133−/CD34− blood cell fraction decreases time from transplantation toneutrophil engraftment in said subject, when compared to transplantationof a single or double unit of unmanipulated cord blood.
 21. (canceled)22. The method of claim 20, wherein said neutrophil engraftment consistsof achieving an absolute neutrophil count (ANC) ≥0.5×109/L on 3consecutive measurements on different days with subsequent donorchimerism (≤10% host cells by peripheral blood chimerism), on or before42 days post transplant.
 23. The method of claim 4, whereintransplantation of said first ex vivo cultured CD133+/CD34+ blood cellfraction and said second uncultured CD133−/CD34− blood cell fractiondecreases time from transplantation to platelet engraftment in saidsubject, when compared to transplantation of a single or double unit ofunmanipulated cord blood.
 24. The method of claim 23, whereintransplantation of said first ex vivo cultured CD133+/CD34+hematopoietic stem/progenitor blood cell fraction and said seconduncultured CD133−/CD34− blood cell fraction increases the probability ofplatelet engraftment in said subject at 42 days post transplantation ordecreases risk of non-engraftment by day 42 after transplantation insaid subject when compared to transplantation of a single or double unitof unmanipulated cord blood.
 25. (canceled)
 26. The method of claim 4,wherein transplantation of said first ex vivo cultured CD133+/CD34+blood cell fraction and said second uncultured CD133−/CD34− blood cellfraction decreases the risk of non-relapse mortality aftertransplantation in said subject, when compared to transplantation of asingle or double unit of unmanipulated cord blood.
 27. The method ofclaim 26, wherein transplantation of said first ex vivo culturedCD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34−blood cell fraction decreases the risk of non-relapse mortality at 210days after transplantation or transplantation related mortality at 1year.
 28. (canceled)
 29. The method of claim 4, wherein transplantationof the first ex vivo cultured CD133+/CD34+ blood cell fraction and saidsecond uncultured CD133−/CD34− blood cell fraction decreases duration ofhospitalization in said subject in the first 100 dayspost-transplantation by 5-30 days, when compared to transplantation of asingle or double unit of unmanipulated cord blood.
 30. (canceled) 31.The method of claim 4, wherein transplantation of the first ex vivocultured CD133+/CD34+ blood cell fraction and said second unculturedCD133−/CD34− blood cell fraction decreases risk of grade 2/3 bacterialor invasive fungal infections post-transplantation in said subject, whencompared to transplantation of a single or double unit of unmanipulatedcord blood.
 32. The method of claim 31, wherein transplantation of thefirst ex vivo cultured CD133+/CD34+ blood cell fraction and said seconduncultured CD133−/CD34− blood cell fraction decreases risk of grade 2/3bacterial or invasive fungal infections by 100 days post-transplantationin said subject, when compared to transplantation of a single or doubleunit of unmanipulated cord blood.
 33. The method of claim 4, whereintransplantation of the first ex vivo cultured CD133+/CD34+ blood cellfraction and said second uncultured CD133−/CD34− blood cell fractiondecreases risk of acute graft-versus-host disease grade III-IV in saidsubject at 100 days post transplantation, when compared totransplantation of a single or double unit of unmanipulated cord blood.34. The method of claim 4, wherein transplantation of the first ex vivocultured CD133+/CD34+ blood cell fraction and said second unculturedCD133−/CD34− blood cell fraction decreases risk of severe chronicgraft-versus-host disease in said subject at 180 days posttransplantation, when compared to transplantation of a single or doubleunit of unmanipulated cord blood.
 35. The method of claim 4, whereinsaid first and second blood cell fractions are co-administered inconjunction with an additional treatment for hematological disease. 36.The method of claim 35, wherein said additional treatment is selectedfrom the group consisting of immunosuppressive treatment, chemotherapyand radio-therapy.
 37. An article of manufacture comprising: (a) apackaging material and (b) umbilical cord blood cell fractionscomprising: (i) a first blood cell fraction suitable for transplantationinto a subject in need thereof comprising CD133+/CD34+ selected cordblood cells ex-vivo cultured under conditions allowing for cellproliferation, said conditions comprising providing nutrients, serum anda combination of cytokines including each of stem cell factor,thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in an amountbetween 1.0 mM to 10 mM; and (ii) a second, uncultured, unselected bloodcell fraction suitable for transplantation into a subject in needthereof comprising CD133/CD34 negative cells, and wherein said packagingmaterial comprises a label or package insert indicating that said firstand second umbilical cord blood cell populations are for treating ahematological disease in a subject in need thereof.
 38. The article ofmanufacture of claim 37, wherein said first ex-vivo cultured blood cellfraction comprises at least 8×108 total viable cells and said seconduncultured blood cell fraction comprises at least 4×108 total viablecells.
 39. (canceled)